Microorganisms for the production of insect pheromones and related compounds

STATEMENT REGARDING THE SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is PRVI_020_01WO_SeqList_ST25.txt. The text file is about 240 KB, was created on May 17, 2018, and is being submitted electronically via EFS-Web.

TECHNICAL FIELD

This application relates to recombinant microorganisms useful in the biosynthesis of unsaturated C₆-C₂₄fatty alcohols, aldehydes, and acetates which may be useful as insect pheromones, fragrances, flavors, and polymer intermediates. The application further relates to methods of producing unsaturated C₆-C₂₄fatty alcohols, aldehydes, and acetates using the recombinant microorganisms, as well as compositions comprising one or more of these compounds and/or the recombinant microorganisms.

BACKGROUND

As the global demand for food grows, there is an increasing need for effective pest control. Conventional insecticides are among the most popular chemical control agents because they are readily available, rapid acting, and highly reliable. However, the overuse, misuse, and abuse of these chemicals have led to resistant pests, alteration of the natural ecology, and in some cases, environmental damage.

The use of insect pheromones to control pest populations has gained increasing popularity as a viable, safe, and environmentally friendly alternative to conventional insecticides. Since their discovery in the late 1950s, these molecules have shown efficacy in reducing insect populations through a variety of methods, including mass trappings, attract and kill, and mating disruption. The latter method in particular represents a non-toxic means of pest control and utilizes the ability of synthetic pheromones to mask naturally occurring pheromones, thereby causing confusion and mating disruption.

Although pheromones have significant potential in agricultural insect control, the cost of synthesizing pheromones using currently available techniques is very high, which prohibits widespread use of this sustainable technology beyond high-value crops. Thus, there is an existing need to develop novel technologies for the cost-efficient production of insect pheromones and related fragrances, flavors, and polymer intermediates. The present inventors address this need with the development of recombinant microorganisms capable of producing a wide-range of unsaturated C₆-C₂₄fatty alcohols, aldehydes, and acetates including synthetic insect pheromones from low-cost feedstocks.

SUMMARY OF THE DISCLOSURE

The present application relates to recombinant microorganisms having a biosynthesis pathway for the production of one or more compounds selected from unsaturated C₆-C₂₄fatty alcohols, aldehydes, and acetates. The recombinant microorganisms described herein may be used for the production of at least one compound, such as an insect pheromone, a fragrance, or a flavoring agent, selected from unsaturated C₆-C₂₄fatty alcohols, aldehydes, and acetates.

In one embodiment, the recombinant microorganism comprises a biosynthesis pathway for the production of an unsaturated C₆-C₂₄fatty aldehyde or fatty alcohol. Accordingly, in a first aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C₆-C₂₄fatty aldehyde or fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acyl-CoA, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding a fatty-acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; and (b): at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty aldehyde. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde is a fragrance or flavoring agent. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acetate. (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is a fragrance or flavoring agent. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty aldehyde. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acetate.

In some embodiments, the fatty-acyl desaturase is a desaturase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

In some embodiments, the fatty-acyl desaturase is capable of generating a double bond at position C5, C6, C7, C8, C9, C10, C11, C12, or C13 in the fatty acid or its derivatives, such as, for example, fatty acid CoA esters.

In one exemplary embodiment, the fatty-acyl desaturase is a Z11 desaturase. In various embodiments described herein, the Z11 desaturase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana. Further Z11-desaturases, or the nucleic acid sequences encoding them, can be isolated from Bombyx mori, Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphania nindalis. In exemplary embodiments, the Z11 desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787, and AY493438. In some embodiments, a nucleic acid sequence encoding a Z11 desaturase from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 9, 18, 24 and 26 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 49 from Trichoplusia ni. In other embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 10 and 16 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 53 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 12, 17 and 30 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transitella. In further embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 13, 19, 25, 27 and 31 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 54 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 39 from S. inferens. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in GenBank Accession nos. AF416738, AGH12217.1, AH21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, a complete or partial Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of a Z11 desaturase is replaced by an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 15, 28 and 29. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 78, 79 and 80.

In certain embodiments, the Z11 desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated product selected from Z11-13:Acyl-CoA, E11-13:Acyl-CoA, (Z,Z)-7,11-13:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,Z)-9,11-15:Acyl-CoA, (Z,Z)-9,11-15:Acyl-CoA, Z11-16:Acyl-CoA, E11-16:Acyl-CoA, (E,Z)-6,11-16:Acyl-CoA, (E,Z)-7,11-16:Acyl-CoA, (E,Z)-8,11-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA. (E,E)-11,13-16:Acyl-CoA, (E,Z)-11,13-16:Acyl-CoA, (Z,E)-11,13-16:Acyl-CoA, (Z,Z)-11,13-16:Acyl-CoA, (Z,E)-11,14-16:Acyl-CoA, (E,E,Z)-4,6,11-16:Acyl-CoA, (Z,Z,E)-7,11,13-16:Acyl-CoA, (E,E,Z,Z)-4,6,11,13-16:Acyl-CoA, Z11-17:Acyl-CoA, (Z,Z)-8,11-17:Acyl-CoA, Z11-18:Acyl-CoA, E11-18:Acyl-CoA, (Z,Z)-11,13-18:Acyl-CoA, (E,E)-11,14-18:Acyl-CoA, or combinations thereof.

In another exemplary embodiment, the fatty-acyl desaturase is a Z9 desaturase. In various embodiments described herein, the Z9 desaturase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Ostrinia furnacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, or Helicoverpa zea. In exemplary embodiments, the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332. AF482906, and AAF81788. In some embodiments, a nucleic acid sequence encoding a Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 20 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 58 from Ostrinia furnacalis. In other embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 60 from Helicoverpa zea. Other Z9 desaturases of the present disclosure include SEQ ID Nos: 95, 97, 99, 101, 103, and 105. In some embodiments, the overexpression of a Z9-18 specific desaturase can increase the membrane fluidity to improve the diffusion of fatty alcohols into the supernatant.

In certain embodiments, the Z9 desaturase catalyzes the conversion of a fatty acyl-CoA into a monounsaturated or polyunsaturated product selected from Z9-11:Acyl-CoA, Z9-12:Acyl-CoA, E9-12:Acyl-CoA, (E,E)-7,9-12:Acyl-CoA, (E,Z)-7,9-12:Acyl-CoA, (Z,E)-7,9-12:Acyl-CoA, (Z,Z)-7.9-12:Acyl-CoA, Z9-13:Acyl-CoA, E9-13:Acyl-CoA, (E,Z)-5,9-13:Acyl-CoA, (Z,E)-5,9-13:Acyl-CoA, (Z,Z)-5.9-13:Acyl-CoA, Z9-14:Acyl-CoA, E9-14:Acyl-CoA, (E,Z)-4,9-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,E)-9,12-14:Acyl-CoA, (Z,E)-9,12-14:Acyl-CoA, (Z,Z)-9,12-14:Acyl-CoA, Z9-15:Acyl-CoA, E9-15:Acyl-CoA, (Z,Z)-6,9-15:Acyl-CoA, Z9-16:Acyl-CoA, E9-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, Z9-17:Acyl-CoA, E9-18:Acyl-CoA, Z9-18:Acyl-CoA, (E,E)-5,9-18:Acyl-CoA, (E,E)-9,12-18:Acyl-CoA, (Z,Z)-9,12-18:Acyl-CoA, (Z,Z,Z)-3,6,9-18:Acyl-CoA, (E,E,E)-9,12,15-18:Acyl-CoA, (Z,Z,Z)-9,12,15-18:Acyl-CoA, or combinations thereof.

In some embodiments, the recombinant microorganism may express a bifunctional desaturase capable of catalyzing the subsequent desaturation of two double bonds.

In some embodiments, the recombinant microorganism may express more than one exogenous nucleic acid molecule encoding a fatty-acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA. For instance, the recombinant microorganism may express an exogenous nucleic acid molecule encoding a Z11 desaturase and another exogenous nucleic acid molecule encoding a Z9 desaturase.

In some embodiments, the recombinant microorganism may express a fatty-acyl conjugase that acts independently or together with a fatty-acyl desaturase to catalyze the conversion of a saturated or monounsaturated fatty acyl-CoA to a conjugated polyunsaturated fatty acyl-CoA.

In one embodiment, the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C₆-C₂₄aldehyde or fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C₆-C₂₄fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl conjugase that catalyzes the conversion of a saturated or monounsaturated C₆-C₂₄fatty acyl-CoA to a corresponding polyunsaturated C₆-C₂₄fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty aldehyde or fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding polyunsaturated C₆-C₂₄fatty aldehyde or fatty alcohol.

In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty-acyl conjugases that catalyze the conversion of a saturated or monounsaturated C₆-C₂₄fatty acyl-CoA to a corresponding polyunsaturated C₆-C₂₄fatty acyl-CoA.

In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C₄-C₂₄fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty-acyl desaturase and at least one exogenous nucleic acid molecule encoding a fatty acyl conjugase that catalyze the conversion of a saturated or monounsaturated C₆-C₂₄fatty acyl-CoA to a corresponding polyunsaturated C₆-C₂₄fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding polyunsaturated C₆-C₂₄fatty alcohol.

In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty-acyl desaturases and at least two exogenous nucleic acid molecules encoding fatty-acyl conjugases that catalyze the conversion of a saturated or monounsaturated C₆-C₂₄fatty acyl-CoA to a corresponding polyunsaturated C₆-C₂₄fatty acyl-CoA.

In yet a further embodiment, the fatty-acyl conjugase is a conjugase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

In certain embodiments, the conjugase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Cydia pomonella, Cydia nigricana, Lobesia botrana, Myelois cribrella, Plodia interpunctella, Dendrolimus punctatus, Lampronia capitella, Spodoptera litura, Amyelois transitella, Manduca sexta, Bombyx mori, Calendula officinalis, Trichosanthes kirilowli, Punica granatum, Momordica charantia, Impatiens balsamina, and Epiphyas postvittana. In exemplary embodiments, the conjugase comprises a sequence selected from GenBank Accession No. or Uniprot database: A0A059TBF5, A0A0M3L9E8, A0A0M3L9S4, A0A0M3LAH8, A0A0M3LAS8, A0A0M3LAH8, B6CBS4, XP_013183656.1, XP_004923568.2, ALA65425.1, NP_001296494.1, NP_001274330.1, Q4A181, Q75PL7, Q9FPP8, AY178444, AY178446, AF182521, AF182520, Q95UJ3.

In various embodiments described herein, the fatty alcohol forming acyl-CoA reductase, i.e., fatty alcohol forming fatty-acyl reductase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Agrotis segetum, Spodoptera littoralis, Helicoverpa amigera, Spodoptera exigua, Euglena gracilis, or Yponomeuta evonymellus. In exemplary embodiments, the reductase comprises a sequence selected from GenBank Accession Nos. JX679210 and HG423128, and UniProt Accession No. I3PN86. In some embodiments, a nucleic acid sequence encoding a fatty-acyl reductase from organisms of the species Agrotis segetum, Spodoptera littoralis, Helicoverpa amigera, Spodoptera exigua, Euglena gracilis, or Yponomeuta evonymellus is codon optimized. In some embodiments, the reductase comprises a nucleotide sequence set forth in SEQ ID NO: 1 from Agrotis segetum. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the reductase comprises a nucleotide sequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the reductase comprises a nucleotide sequence selected from SEQ ID NOs: 3, 32, 40, 72, 74, 76 and 81. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 77 from Yponomeuta evonymellus.

In some embodiments, the present disclosure teaches using multiple fatty acyl reductase enzymes. In some embodiments, the present disclosure teaches recombinant microorganisms comprising multiple copies of the same fatty acyl reductase. In other embodiments, the present disclosure teaches recombinant microorganisms comprising two or more different fatty acyl reductases. In some embodiments, the different fatty acyl reductases utilize different co-factors. For example, the fatty acyl reductase from Euglena gracilis (SEQ ID NO: 75) uses NADH instead of NADPH as reducing equivalent. In some embodiments, this can allow for co-factor balancing using two or more different reductases.

In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42-48. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises a nucleotide sequence selected from SEQ ID NOs: 83-89.

In certain embodiments, the fatty alcohol forming fatty-acyl reductase catalyzes the conversion of a mono- or poly-unsaturated fatty acyl-CoA into a fatty alcohol product selected from (Z)-3-hexenol, (Z)-3-nonenol, (Z)-5-decenol, (E)-5-decenol, (Z)-7-dodecenol, (E)-7-dodecenol, (E)-8-dodecenol, (Z)-8-dodecenol, (Z)-9-dodecenol, (E)-9-dodecenol, (Z)-9-tetradecenol, (E)-9-tetradecenol, (Z)-9-hexadecenol, (Z)-11-tetradecenol, (Z)-7-hexadecenol, (Z)-11-hexadecenol, (E)-11-hexadecenol (E)-11-tetradecenol, or (Z,Z)-11,13-hexadecadienol, (11Z,13E)-hexadecadienol, (E,E)-8,10-dodecadienol, (E,Z)-7,9-dodecadienol, (Z)-13-octadecenol, or combinations thereof.

In some embodiments, the recombinant microorganism may express more than one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of a mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.

In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C₄-C₂₂fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA substrate in that cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C₁₈fatty alcohol. In some embodiments, the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lamproninia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase. In some embodiments, the fatty acyl desaturase has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85% 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AH21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the acyl-CoA oxidase is selected from Table 5a. In other embodiments, the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase. In further embodiments, the fatty alcohol forming fatty acyl reductase has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82. In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.

In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C₁₈fatty acyl-CoA. In some embodiments, the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT). In some preferred embodiments, the acyltransferase is selected from Table 5b.

In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates. In some embodiments, the acylglycerol lipase is selected from Table 5c.

In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ≤C₁₈fatty alcohol. In further embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from: (i) one or more acyl-CoA oxidase; (ii) one or more acyltransferase; (iii) one or more acylglycerol lipase and/or sterol ester esterase; (iv) one or more (fatty) alcohol dehydrogenase; (v) one or more (fatty) alcohol oxidase; and (vi) one or more cytochrome P450 monooxygenase.

In some preferred embodiments, one or more genes of the microbial host encoding acyl-CoA oxidases are deleted or down-regulated to eliminate or reduce the truncation of desired fatty acyl-CoAs beyond a desired chain-length. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y. lipolytica POX1 (YALI0E32835g), Y. lipolytica POX2 (YALI0F10857g). Y. lipolytica POX3 (YALI0D24750g), Y. lipolytica POX4 (YALI0E27654g), Y. lipolytica POX5 (YALI0C23859g), Y. lipolytica POX6 (YALI0E06567g); S. cerevisiae POX1 (YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).

In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750. Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.

In some preferred embodiments, one or more genes of the microbial host encoding acylglycerol lipases (mono-, di-, or triacylglycerol lipases) and sterol ester esterases are deleted or downregulated and replaced with one or more acylglycerol lipases which prefer long chain acylglycerol substrates. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864. Candida CaO19.12328. Candida CTRG_03360, Candida CaO19.6501. Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.

In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6), Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK12).

In some embodiments, the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA. E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z5-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA, E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E14-16:Acyl-CoA, Z13E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z11Z13-16:Acyl-CoA, and Z13Z15-18:Acyl-CoA. In further embodiments, the mono- or poly-unsaturated ≤C₁₈fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH. E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH, and Z13-18:OH.

In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty acid into a corresponding ≤C₁₈fatty aldehyde. In some preferred embodiments, the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A117N158, and Nostoc punctiforme YP_001865324. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde. In some preferred embodiments, the ≤C₁₈fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.

In some embodiments, the recombinant microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty acetate. In some preferred embodiments, the mono- or poly-unsaturated ≤C₁₈fatty aldehyde and ≤C₁₈fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.

In some embodiments, the disclosure provides a method of engineering a microorganism that is capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the method comprises introducing into a microorganism the following: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C18 fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C₄-C₂₂fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA substrate in that cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C₁₈fatty alcohol. In some embodiments, the microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.

In some embodiments, the disclosure provides a method of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde or fatty acetate. In some embodiments, the method further comprises a step of recovering the mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde or fatty acetate. In further embodiments, the recovery step comprises distillation. In yet further embodiments, the recovery step comprises membrane-based separation.

In some embodiments, the mono- or poly-unsaturated ≤C₁₈fatty alcohol is converted into a corresponding ≤C₁₈fatty aldehyde using chemical methods. In further embodiments, the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)₂, Swern oxidation and noble metal-air. In some embodiments, the mono- or poly-unsaturated ≤C₁₈fatty alcohol is converted into a corresponding ≤C₁₈fatty acetate using chemical methods. In further embodiments, the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated ≤C₁₈fatty alcohol to the corresponding ≤C₁₈fatty acetate.

In a further embodiment, the disclosure provides a recombinant Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the recombinant Yarrowia lipolytica microorganism comprises: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.

In some embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3). YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12): YAS1 (YALI1C03349), Yas2 (YALI0E32417), Gsyl1 (YALI0F18502), HFD1 (YALI0F23793), HFD2 (YALI0E15400), HFD3 (YALI0A17875), HFD4 (YALI0B01298), SDR (YALI0A19536); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2). In other preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F01857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10). YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).

In some embodiments, the fatty acyl desaturase catalyzes the conversion of a saturated fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from Z9-14:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, Z9-16:Acyl-CoA, and Z11-16:Acyl-CoA. In other embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.

In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde. In some embodiments, the alcohol dehydrogenase is selected from Table 3a. In some embodiments, the C₆-C₂₄fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde and C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z1Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

In some embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66 and 67. In other embodiments, the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from an Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or Trichoplusia ni derived desaturase.

In some embodiments, the disclosure provides a method of engineering a Yarrowia lipolytica microorganism that is capable of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the method comprises introducing into the Yarrowia lipolytica microorganism the following: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%& 61%, 60&% 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 55, 56, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some embodiments, the microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.

In some embodiments, the disclosure provides a method of producing a mono- or poly-unsaturated C₆-C₄fatty alcohol, fatty aldehyde or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol, fatty aldehyde or fatty acetate. In some embodiments, the method further comprises a step of recovering the mono- or poly-unsaturated C₆-C₂₄fatty alcohol, fatty aldehyde or fatty acetate. In further embodiments, the recovery step comprises distillation. In yet further embodiments, the recovery step comprises membrane-based separation.

In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is converted into a corresponding C₆-C₂₄fatty aldehyde using chemical methods. In further embodiments, the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)₂, Swern oxidation and noble metal-air. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is converted into a corresponding C₆-C₂₄fatty acetate using chemical methods. In further embodiments, the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N. N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated C₆-C₂₄fatty alcohol to the corresponding C₆-C₂₄fatty acetate.

In addition to the biosynthetic pathway described in the first aspect above, the present application provides an additional biosynthetic pathway for the production of an unsaturated C₆-C₂₄fatty alcohol utilizing a saturated C₆-C₂₄fatty acyl-ACP intermediate derived from a C₆-C₂₄fatty acid. Accordingly, in a second aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of C₆-C₂₄fatty acid, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of a C₆-C₂₄fatty acid to a corresponding saturated C₆-C₂₄fatty acyl-ACP; (b) at least one exogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-ACP to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP; (c) one or more endogenous or exogenous nucleic acid molecules encoding a fatty acid synthase complex that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP from (b) to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP with a two carbon elongation relative to the product of (b); (d): at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP from (c) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty aldehyde; and (e) at least one endogenous or exogenous nucleic acid molecule encoding a dehydrogenase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde C₆-C₂₄from (d) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is a fragrance or flavoring agent. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty aldehyde. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acetate.

In some embodiments, acyl-ACP synthetase is a synthetase capable of utilizing a fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

In various embodiments described herein, the acyl-ACP synthetase, or the nucleic acid that encodes it, can be isolated from organisms of the species Vibrio harveyi, Rhodotorula gluntnis, or Yarrowia lipolytica.

In some embodiments, the fatty-acyl-ACP desaturase is a soluble desaturase. In various embodiments described herein, the fatty-acyl-ACP desaturase, or the nucleic acid that encodes it, can be isolated from organisms of the species Pelargonium hortorum, Asclepias syriaca, or Uncaria tomentosa.

As described above, fatty acid elongation enzymes, i.e., a fatty acid synthase complex, can be utilized to extend the chain length of a mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP by two additional carbons at the alpha carbon. In some embodiments, the two additional carbons are derived from endogenous malonyl-CoA. In one embodiment, the one or more nucleic acid molecules encoding a fatty acid synthase complex are endogenous nucleic acid molecules, i.e., the nucleic acid molecule(s) is/are native to the recombinant microorganism. In another embodiment, the one or more nucleic acid molecules encoding a fatty acid synthase complex are exogenous nucleic acid molecules.

In various embodiments described herein, the fatty aldehyde forming acyl-ACP reductase, i.e., fatty aldehyde forming fatty-acyl reductase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species can be isolated from organisms of the species Pelargonium hortorum, Asclepias syriaca, and Uncaria tomentosa.

As noted above, the recombinant microorganism according to the second aspect comprises at least one endogenous or exogenous nucleic acid molecule encoding a dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde from (d) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In one embodiment, the dehydrogenase is encoded by an endogenous nucleic acid molecule. In another embodiment, the dehydrogenase is encoded by an exogenous nucleic acid molecule. In exemplary embodiments, the endogenous or exogenous nucleic acid molecule encoding a dehydrogenase is isolated from organisms of the species Saccharomyces cerevisiae, Escherichia coli, Yarrowia lipolytica, or Candida tropicalis.

In addition to the biosynthetic pathway described in the first and second aspects above, the present application provides an additional biosynthetic pathway for the production of an unsaturated C₆-C₂₄fatty alcohol utilizing a saturated C₆-C₂₄fatty acyl-ACP intermediate derived from a C₆-C₂₄fatty acid. Accordingly, in a third aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of C₆-C₂₄fatty acid, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of a C₆-C₂₄fatty acid to a corresponding saturated C₆-C₂₄fatty acyl-ACP; (b) at least one exogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-ACP to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP; (c) at least one exogenous fatty acyl-ACP thioesterase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP from (b) to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acid; (d) one or more endogenous or exogenous nucleic acid molecules encoding an elongase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA derived from CoA activation of the mono- or poly-unsaturated C₆-C₂₄fatty acid from (c) to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA with a two carbon or greater elongation relative to the product of (c); and (e): at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (d) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is an insect pheromone. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is a fragrance or flavoring agent. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty aldehyde. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol from (e) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acetate.

In some embodiments according to this third aspect, a fatty acyl-ACP thioesterase can be utilized to convert a mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acid. In a some embodiments, soluble fatty acyl-ACP thioesterases can be used to release free fatty acids for reactivation to a CoA thioester. Fatty acyl-ACP thioesterases that can be included within the embodiment include, but are not limited to, including Q41635, Q39473, P05521.2, AEM72519, AEM72520, AEM72521, AEM72523, AAC49784, CAB60830, EER87824, EER96252, ABN54268, AAO77182, CAH09236, ACL08376, and homologs thereof may be used. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA may serve as a substrate for an elongase, which can be utilized to extend the chain length of a mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA by two additional carbons at the alpha carbon. In some embodiments, the two additional carbons are derived from endogenous malonyl-CoA.

As described above, in some embodiments, the recombinant microorganism according to the first, second, or third aspect further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase capable of catalyzing the conversion of a mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty aldehyde. In certain embodiments, the alcohol oxidase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Candida boidinii, Komagataella pastoris, Tanacetum vulgare, Simmondsia chinensis, Arabidopsis thaliana, Lotus japonicas, or Candida tropicalis. In exemplary embodiments, the alcohol oxidase comprises a sequence selected from GenBank Accession Nos. Q00922, F2QY27, Q6QIR6, Q8LDP0, and L7VFV2.

As described above, in some embodiments, the recombinant microorganism according to the first or second aspect further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of a C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate. In certain embodiments, the acetyl transferase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Saccharomyces cerevisiae, Danaus plexippus, Heliotis virescens, Bombyx mori, Agrotis ipsilon, Agrotis segetum, Euonymus alatus. In exemplary embodiments, the acetyl transferase comprises a sequence selected from GenBank Accession Nos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, XP_505595.1, and XP_505513.1.

In alternative embodiments, the fatty alcohol may be converted into a fatty acetate using chemical methods, e.g., via chemical catalysis utilizing a chemical agent such as acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride.

In some embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate may further be engineered to express one or more nucleic acids encoding protein or polypeptide which, when expressed, is toxic to an insect. Exemplary toxicant producing genes suitable for the present disclosure can be obtained from entomopathogenic organism, such as Bacillus thuringiensis. Pseudomonas aeruginosa, Serratia marcescens, and members of the genus Streptomyces. In an exemplary embodiment, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate may further be engineered to express a nucleic acid encoding a Bacillus thuringiensis (“Bt”) toxin. In additional or alternative embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate may further be engineered to express a nucleic acid encoding other toxic proteins such as spider venom.

In some embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate may further be engineered to express a metabolic pathway which, when expressed, produces a small molecule that is toxic to an insect. Non-limiting examples of toxic small molecules include azadirachtin, spinosad, avermectin, pyrethrins, and various terpenoids.

In various embodiments described herein, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate may be a eukaryotic microorganism, such as a yeast, a filamentous fungi, or an algae, or alternatively, a prokaryotic microorganism, such as a bacterium. For instance, suitable host cells can include cells of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, and Streptomyces.

In some embodiments, the recombinant microorganism comprising a biosynthesis pathway for the production of an unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate is a yeast. Examples of suitable yeasts include yeasts of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluveromyces, Issatchenkia, Zygosaccharomyes, Debaryomyces, Schizosaccharomyces Pachysolen, Cyptococcus, Trichosporon, Rhodotorula, or Myxozyma. In certain embodiments, the yeast is an oleaginous yeast. Exemplary oleaginous yeasts suitable for use in the present disclosure include members of the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces, including, but not limited to the species of Yarrowia lipolytica, Candida tropicalis, Rhodosporidium toruloides, Lipomyces starkey, L. lipoferus, C. revkaufi, C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T. cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.

As will be understood in the art, endogenous enzymes can convert critical substrates and/or intermediates upstream of or within the unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate biosynthesis pathway into unwanted by-products. Accordingly, in some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate biosynthesis pathway.

In one embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acid into a ω-hydroxyfatty acid. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from XP_504406, XP_504857, XP_504311, XP_500855, XP_500856, XP_500402, XP_500097, XP_501748, XP_500560, XP_501148, XP_501667, XP_500273, BAA02041, CAA39366, CAA39367, BAA02210, BAA02211, BAA02212, BAA02213, BAA02214, AAO73952, AAO73953, AAO73954, AAO73955, AAO73956, AAO73958, AAO73959, AAO73960, AAO73961, AAO73957, XP_002546278, or homologs thereof. In the context of a recombinant bacterial microorganism, the recombinant bacterial microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from BAM49649, AAB80867, AAB17462, ADL27534, AAU24352, AAA87602, CAA34612, ABM17701, AAA25760, CABS 1047, AAC82967, WP_011027348, or homologs thereof.

In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acyl-CoA into α,β-enoyl-CoA. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, CAG79214, AAA34322, AAA34361, AAA34363, CAA29901, BAA04761, AAA34891, or homologs thereof. In the context of a recombinant bacterial microorganism, the recombinant bacterial microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from AAB08643, CAB15271, BAN55749, CAC44516, ADK16968, AEI37634, WP_000973047, WP_025433422, WP_035184107, WP_026484842, CEL80920, WP_026818657, WP_005293707, WP_005883960, or homologs thereof.

In embodiments where the recombinant microorganism is a yeast microorganism, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme involved in peroxisome assembly and/or peroxisome enzyme import. The recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more enzyme selected from XP_505754, XP_501986, XP_501311, XP_504845, XP_503326, XP_504029, XP_002549868, XP_002547156, XP_002545227, XP_002547350, XP_002546990, EIW11539, EIW08094, EIW11472, EIW09743, EIW08286, or homologs thereof.

In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous reductase or desaturase enzymes that interferes with the unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate, i.e., catalyzes the conversion of a pathway substrate or product into an unwanted by-product.

In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous alcohol oxidase or alcohol dehydrogenase enzymes that catalyzes the unwanted conversion of the desired product, e.g., unsaturated C₆-C₂₄fatty alcohol into a corresponding unsaturated C₆-C₂₄fatty aldehyde.

In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for one or more unsaturated fatty acyl-CoA intermediates. In one embodiment, the one or more endogenous enzymes comprise one or more diacylglycerol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more diacylglycerol acyltransferases selected from the group consisting of YALI0E32769g, YALI0D07986g and CTRG_06209, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more glycerolphospholipid acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more glycerolphospholipid acyltransferases selected from the group consisting of YALI0E16797g and CTG_04390, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more acyl-CoA/sterol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more acyl-CoA/sterol acyltransferases selected from the group consisting of YALI0F06578g, CTRG_01764 and CTRG_01765, or homolog thereof.

In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that consumes fatty acetate products. In one embodiment, the one or more endogenous enzymes comprise one or more sterol esterases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more sterol esterases selected from the group consisting of YALI0E32035g, YALI0E00528g, CTRG_01138, CTRG_01683 and CTRG_04630, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more triacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more triacylglycerol lipases selected from the group consisting of YALI0D17534g, YALI0F10010g, CTRG_00057 and CTRG_06185, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more monoacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more monoacylglycerol lipases selected from the group consisting of YALI0C14520g, CTRG_03360 and CTRG_05049, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more extracellular lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more extracellular lipases selected from the group consisting of YALI0A20350g, YALI0D19184g, YALI0B09361g, CTRG_05930, CTRG_04188, CTRG_02799, CTRG_03052 and CTRG_03885, or homolog thereof.

In embodiments where the recombinant microorganism is a yeast microorganism, one or more of the exogenous unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate pathway genes encodes an enzyme that is localized to a yeast compartment selected from the group consisting of the cytosol, the mitochondria, or the endoplasmic reticulum. In an exemplary embodiment, one or more of the exogenous pathway genes encodes an enzyme that is localized to the endoplasmic reticulum. In another embodiment, at least two exogenous pathway genes encode an enzyme that is localized to the endoplasmic reticulum. In yet another embodiment, all exogenous pathway genes encodes an enzyme that is localized to the endoplasmic reticulum.

In additional embodiments, the present application provides methods of producing an unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate using a recombinant microorganism as described herein. In one embodiment, the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until the unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate is produced and optionally, recovering the unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate. Once produced, the unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate may be isolated from the fermentation medium using various methods known in the art including, but not limited to, distillation, membrane-based separation gas stripping, solvent extraction, and expanded bed adsorption.

In some embodiments, the recombinant microorganism, e.g., a yeast, may be recovered and produced in dry particulate form. In embodiments involving yeast, the yeast may be dried to produce powdered yeast. In some embodiments, the process for producing powdered yeast comprises spray drying a liquid yeast composition in air, optionally followed by further drying. In some embodiments, the recombinant microorganism composition will comprise the unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate when dried.

As described herein, preferred recombinant microorganisms of the disclosure will have the ability to utilize alkanes and fatty acids as carbon sources. However, as will be understood in the art, a variety of carbon sources may be utilized, including but not limited to, various sugars (e.g., glucose, fructose, or sucrose), glycerol, alcohols (e.g., ethanol), organic acids, lignocellulose, proteins, carbon dioxide, carbon monoxide, as well as the aforementioned alkanes and fatty acids. In some embodiments, the recombinant microorganism will convert the carbon source to the unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate under aerobic conditions.

As highlighted above, the present application provides methods of producing one or more unsaturated C₆-C₂₄fatty alcohols, aldehydes, or acetates using a recombinant microorganism as described herein. In some embodiments, the product is an insect pheromone. As will be appreciated by the skilled artisan equipped with the instant disclosure, a variety of different exogenous and endogenous enzymes can be expressed in a recombinant host microorganism to produce a desired insect pheromone. Exemplary insect pheromones in the form of fatty alcohols, fatty aldehydes, or fatty acetates capable of being generated using the recombinant microorganisms and methods described herein include, but are not limited to, (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, (Z)-9-tetradecenyl acetate, (Z,Z)-11,13-hexadecadienal, (9Z,11E)-hexadecadienal, (E,E)-8,10-dodecadien-1-ol, (7E,9Z)-dodecadienyl acetate, (Z)-3-nonen-1-ol, (Z)-5-decen-1-ol, (Z)-5-decenyl acetate, (E)-5-decen-1-ol, (E)-5-decenyl acetate, (Z)-7-dodecen-1-ol, (Z)-7-dodecenyl acetate, (E)-8-dodecen-1-ol, (E)-8-dodecenyl acetate, (Z)-8-dodecen-1-ol, (Z)-8-dodecenyl acetate, (Z)-9-dodecen-1-ol, (Z)-9-dodecenyl acetate, (Z)-9-tetradecen-1-ol, (Z)-11-tetraceden-1-ol, (Z)-11-tetracedenyl acetate, (E)-11-tetradecen-1-ol, (E)-11-tetradecenyl acetate, (Z)-7-hexadecen-1-ol, (Z)-7-hexadecenal, (Z)-9-hexadecen-1-ol, (Z)-9-hexadecenal, (Z)-9-hexadecenyl acetate, (Z)-11-hexadecen-1-ol, (Z)-13-octadecen-1-ol, (Z)-13-hexadecenyl acetate, and (Z)-13-octadecenayl acetate, and (Z)-13-octadecenal.

In another embodiment of the present application, compositions comprising one of more of the insect pheromone-producing recombinant microorganisms described herein can be provided. In certain embodiments, the composition may further comprise one or more insect pheromones produced by the recombinant microorganism. In further embodiments, the may additionally comprise one or more toxic proteins or polypeptides produced by the recombinant microorganism.

BRIEF DESCRIPTION OF DRAWINGS

Illustrative embodiments of the disclosure are illustrated in the drawings, in which:

FIG. 1 illustrates the conversion of a saturated fatty acyl-CoA to an unsaturated fatty alcohol.

FIG. 2 illustrates the conversion of a saturated fatty acid to a mono- or poly-unsaturated fatty aldehyde, alcohol, or acetate.

FIG. 3 illustrates an additional pathway for the conversion of a saturated fatty acid to a mono- or poly-unsaturated fatty aldehyde, alcohol, or acetate.

FIG. 4 illustrates a pathway for the conversion of a saturated fatty acid to various trienes, dienes, epoxides, and odd-numbered pheromones.

FIG. 5 shows Z11-hexadecenol production from W303A and BY4742 ΔPOX1. Strain expressing empty vector (EV), S. littoralis reductase (FAR-SL), H. armigera reductase (FAR-HA), A. segetum reductase (FAR-AS). Error bars represent standard deviation derived from N=2 biologically independent samples.

FIG. 6A-FIG. 6B shows sample chromatograms of biotransformation product of Z11-hexadecenoic acid using S. cerevisiae expressing either an empty vector (FIG. 6A), or Helicoverpa armigera alcohol-forming reductase (FIG. 6B). Black lines: no substrate added. Purple line: Z11-hexadecenoic acid was added as substrate.

FIG. 7A-FIG. 7B shows a comparison of GC-MS fragmentation pattern of Z11-hexadecenol authentic compound (FIG. 7A), and Z11-hexadecenol biologically derived (FIG. 7B).

FIG. 8 shows biomass at the time of harvesting for product analysis of W303A (wild type) and BY4742 ΔPOX1 (beta-oxidation deletion mutant). Strain expressing empty vector (EV), S. littoralis reductase (FAR-SL), H. armigera reductase (FAR-HA), A. segetum reductase (FAR-AS). Error bars represent standard deviation derived from N=2 biologically independent samples.

FIG. 9 shows a Z11-hexedecenol calibration curve constructed using an authentic standard. The samples were generated with the extraction and analysis method described in Materials and Methods of Example 3. Error bars represent standard deviation derived from N=3 samples.

FIG. 10 shows a pOLE1 cassette comprising an extended OLE1 promoter sequence (light yellow), OLE1 promoter (orange), OLE1 leader sequence (dark grey), a synthon such as an insect desaturase sequence (light grey), and the VSP13 terminator sequence (blue).

FIG. 11A-FIG. 11E shows validation of the pOLE1 cassette, and complementation assay. FIG. 11A: YPD+palmitoleic acid; FIG. 11B: YPD−palmitoleic acid; FIG. 11C: CM-Ura glucose+palmitoleic acid; FIG. 11D: CM-Ura glucose—palmitoleic acid, FIG. 11E: Map of strains in FIG. 11A-FIG. 11D. Dasher=GFP synthon.

FIG. 12A shows complementation of ΔOLE1 growth without UFA on YPD.

FIG. 12B shows complementation of ΔOLE1 growth without UFA on CM-Ura glucose.

FIG. 13A shows the full fatty acid spectrum of a ΔOLE1 strain expressing: S. cerevisiae OLE desaturase (blue), chimeric T. ni desaturase (red).

FIG. 13B shows a focused fatty acid spectrum within 5.5-min-8-min retention time of S. cerevisiae ΔOLE1 strain expressing S. cerevisiae OLE1 desaturase (red) and chimeric T. ni desaturase (blue).

FIG. 14A-FIG. 14B shows a comparison of GC-MS fragmentation pattern of (Z)-11-hexadecenoic acid from an authentic compound (FIG. 14A) and biologically derived (FIG. 14B).

FIG. 15 shows C16 fatty alcohol production from ΔOLE1 expressing various fatty alcohol pathway variants in culture supplemented with palmitic and palmitoleic acid. Error bars represent 5% uncertainty of metabolite quantification accuracy.

FIG. 16 shows representative chromatograms of biotransformation product C16 fatty acids using S. cerevisiae expressing fatty alcohol pathways TN_desat-HA_reduc when fed with palmitic acid (black) and when fed with palmitic and palmitoleic acids (orange). Profile of a negative control strain (harboring an empty vector) fed with palmitic acid (purple).

FIG. 17 shows that (Z)-11-hexadecenoic acid was detected in the cell pellets of S. cerevisiae expressing fatty alcohol pathways TN_desat-SL_reduc (blue), SC_desat-HA_reduc (red), TN_desat-HA_reduc (green). SC_desat-SL_reduc (pink).

FIG. 18 shows C16 fatty alcohol production from ΔOLE1 expressing various fatty alcohol pathway variants in culture supplemented with palmitic acid only. Error bars represent 5% uncertainty of metabolite quantification accuracy.

FIG. 19A-FIG. 19C shows detection of (Z)-11-hexadecenol. FIG. 19A: Fragmentation pattern of an authentic standard. The m/z 297.3 was used in follow up experiments to selectively detect the alcohol. To also detect the internal standard, the masses 208 and 387.3 were included too. FIG. 19B: In addition to the detection of the specific mass fragment, the retention time was used as second stage confirmation. The retention time is 6.22. FIG. 19C: Comparison of the two different regioisomers 9Z- and 11Z-hexadcenol when detected in SIM mode (297.3) with the same method.

FIG. 20 shows pXICL expression cassette architecture. The C. albicans OLE1 leader-A. segetum desaturase fusion is also shown.

FIG. 21A-FIG. 21D shows mCherry control integration. FIG. 21A: Negative (water-only) control transformation plate. FIG. 21B: pPV0137 mCherry transformation plate. FIG. 21C: Patch plates from negative control clones. FIG. 21D: Patch plates from pPV0137 clones.

FIG. 22 shows integration efficiency as a function of total observed colonies. A control plate with no DNA added to the transformation was observed to have 350 colonies (indicated by orange line). The fraction of clones confirmed to be positive integrants is positively correlated with total colony count. A sharp increase is observed above 6,000 total colonies. The data suggests that the presence of positive integrants increases the observed background growth. For some transformations the efficiency was high enough that the background population was small relative to the positive integrant population.

FIG. 23 shows a chromatogram overlay of Candida tropicalis SPV053 strains. Compared to the mCherry (red) control experiment a clear peak at 6.22 min is observable for the A. transitella (blue) and H. zea (green) desaturase. Therefore, the formation of Z-11-hexadecenoic acid is only observable in strains expressing an active Z11-desaturase.

FIG. 24A-FIG. 24E shows confirmation of the 11Z-regioisomer. FIG. 24A: The specific peak with an ion fragment of 245 m/z was only observed in C. tropicalis SPV053 expressing either the Z11-desaturase from A. transitella or H. zea. FIG. 24B: The fragmentation patterns of the authentic standard. FIG. 24D: The fragmentation patterns of the newly formed compound in samples with expressed desaturase from H. zea match those of the standard. FIG. 24E: The fragmentation patterns of the newly formed compound in samples with expressed desaturase from A. transitella match those of the standard. FIG. 24C: The fragmentation patterns of the mCherry control significantly differ from those of FIG. 24B. FIG. 24D and FIG. 24E.

FIG. 25A-FIG. 25B shows a GC-FID chromatogram of different C. tropicalis SPV053 strains incubated with methyl tretradecanoate. FIG. 25A: Overall spectrum. The occurrence of the Z11-C16:1 peak is observable for the strains expressing the Z11-desaturases from A. transitella and H. zea. FIG. 25B: Zoom of the C14 to C18 area. A new peak is visible at 4.8 min, which could correspond to Z11-C14:1. Another peak near Z9-C18:1 is also visible, which could correspond to Z11-C18:1.

FIG. 26 shows only codon optimized H. zea desaturase variants produce detectable Z11-hexadecenoic acid in SPV140 screen, control=pPV101 integrants of SPV140, T. ni native=T. ni Z11 desaturase with native codon usage (pPV195), T. ni HS opt=T. ni Z11 desaturase with Homo sapiens codon optimization (pPV196), T. ni HS opt Y1 leader=T. ni Z11 desaturase with Homo sapiens codon optimization and swapped Y. lipolytica OLE1 leader sequence (pPV197), H. zea native=H. zea Z11 desaturase with native codon usage (pPV198), H. zea HS opt=H. zea Z11 desaturase with Homo sapiens codon optimization (pPV199), H. zea HS opt Y1 leader=H. zea Z11 desaturase with Homo sapiens codon optimization and swapped Y. lipolytica OLE1 leader sequence (pPV200). A. transitella native=A. transitella Z11 desaturase with native codon usage (pPV201). All data average of 3 biological replicates. Error bars represent standard deviation.

FIG. 27 shows only codon optimized H. zea desaturase variants produce detectable Z11-hexadecenoic acid in SPV300 screen. Labels indicate parent strain and plasmid of desaturase expression cassette, pPV101=hrGFP control, pPV198=H. zea Z11 desaturase with native codon usage, pPV199=H. zea Z11 desaturase with Homo sapiens codon optimization, pPV200=H. zea Z11 desaturase with Homo sapiens codon optimization and swapped Y. lipolytica OLE1 leader sequence, pPV201=A. transitella Z11 desaturase with native codon usage.

FIG. 28 shows final cell densities for desaturase screen in SPV140 and SPV300 backgrounds. SPV300 strains with integrated desaturase cassettes grew to higher cell densities.

FIG. 29 shows individual isolate Z11-hexadecenoic acid titers for SPV140 and SPV300 strains expressing H. zea Z11 desaturase with H. sapiens codon optimization.

FIG. 30 shows a chromatogram overlay of extracted metabolites for Z11-16OH producing strain (SPV0490) versus control strain (SPV0488) of Candida viswanathii (tropicalis).

FIG. 31 illustrates pathways that can be deleted or disrupted to reduce or eliminate competition with the biosynthesis pathway for the production of a mono- or poly-unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate.

FIG. 32A-FIG. 32B shows Z9-16OH and Z11-16OH titers in YPD (FIG. 32A) and Semi-Defined C:N=80 (FIG. 32B) media for pEXP clones. Ten isolates expressing the H. zea desaturase under the TEF promoter and H. armigera reductase under the EXP promoter from two independent competent cell preparations (Comp. Cell Preparation 1, Comp. Cell Preparation 2) were compared to a parental negative control (SPV300) and a desaturase only negative control (SPV459 Hz_desat only). Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2). *One replicate from Clone 5 and Clone 18 under the Semi-Defined C:N=80 condition was lost during sample work-up so the titers for that condition are from a single data point (N=1, Comp. Cell Preparation 1 Clone 18 and Comp. Cell Preparation 2 Clone 5).

FIG. 33A-FIG. 33B shows profiles of 16-carbon fatty acid species in YPD (FIG. 33A) and Semi-Defined C:N=80 (FIG. 33B) media for pEXP1 clones. The 16-carbon lipid profiles of 5 select clones expressing the H. zea desaturase under the TEF promoter and H. armigera reductase under the EXP promoter are compared to a parental negative control (SPV300) and a desaturase only negative control (SPV459 Hz_desat only). Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2).

FIG. 34 shows Z9-16OH and Z11-16OH titers in Semi-Defined C:N=80 media for pTAL1 clones. Nine isolates expressing the H. zea desaturase under the TEF promoter and H. armigera reductase under the TAL promoter were compared to a parental negative control (SPV300) and positive Bdr pathway controls using the EXP promoter to drive H. armigera FAR expression (SPV575, SPV578). Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2).

FIG. 35 shows profiles of 16-carbon fatty acid species in Semi-Defined C:N=80 medium for pTAL1 clones. The 16-carbon lipid profiles of 5 select clones expressing the H. zea desaturase under the TEF promoter and H. armigera reductase under the EXP promoter are compared to a parental negative control (SPV300) and positive Bdr pathway controls using the EXP promoter to drive H. armigera FAR expression (SPV575, SPV578). Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2). * indicates clones for which one of the replicates was lost during sample processing, N=1.

FIG. 36 shows full Bdr pathway pTAL1 screen (strains expressing H. zea Z11 desaturase (pTEF) and H. armigera FAR) full lipid profiles in Semi-Defined C:N=80 medium after 48 hours of bioconversion. Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2). * indicates clones for which one of the replicates was lost during sample processing, N=1.

FIG. 37A-FIG. 37B shows SPV471 (H222 ΔPΔAΔF expressing native Y. lipolytica OLE1 and H. armigera FAR) Z9-16OH (FIG. 37A) and fatty acid (FIG. 37B) titers in Semi-Defined C:N=80 medium after 24 hours of bioconversion. Error bars represent the SEM (standard error of the mean) measured from technical replicates for each strain and condition (N=2).

FIG. 38 shows SPV471 (H222 ΔPΔAΔF expressing native Y. lipolytica OLE1 and H. armigera FAR) full lipid profiles in Semi-Defined C:N=80 medium after 24 hours of bioconversion.

FIG. 39A-FIG. 39B shows SPV471 (H222 ΔPΔAΔF expressing native Y. lipolytica OLE1 and H. armigera FAR) Z9-16OH (FIG. 39A) and Z9-16 Acid (FIG. 39B) titer time courses. Bioconversion of 16Acid was conducted in Semi-Defined C:N=80 medium using a methyl palmitate (16Acid) substrate.

FIG. 40 shows examples of acyl-CoA intermediates generated through selective β-oxidation controlled by acyl-CoA oxidase activity.

FIG. 41 shows Z11-14Acid (methyl myristate fed—14ME) and Z11-16Acid (methyl palmitate fed—16ME) titers of characterized Δ11 desaturases. SPV300=desaturase library integration parent. SPV298=prototrophic parent of SPV300, negative control. SPV459=SPV300 with current best desaturase (Helicoverpa zea, SEQ ID NO: 54), positive control. The desaturase in DST006 is genetically equivalent to the H. zea desaturase expressed in SPV459 and served as an internal library control.

FIG. 42 shows C14 and C18 product profiles of SPV298 (negative control, parent strain) and SPV459 (SPV298 lineage with H. zea desaturase, SEQ ID NO: 54) fed on either methyl palmitate (16ME) or methyl myristate (14ME).

FIG. 43 shows bioinformatic analysis of potential serine, threonine and tyrosine phosphorylation sites of the H. amigera FAR enzyme (SEQ ID NO: 41). The horizontal line resembles the threshold for potential phosphorylation.

FIG. 44 shows bioinformatic analysis of potential serine and threonine phosphorylation sites of the Helicoverpa amigera derived FAR enzyme upon expression in yeast. The used server (world wide web address: cbs.dtu.dk/services/NetPhosYeast/; Blom, N., Gammeltoft, S. & Brunak, S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites1. J. Mol. Biol. 294, 1351-1362 (1999)) predicts phosphorylated amino acids specifically in yeast. The horizontal line resembles the threshold for possible phosphorylation sites.

FIG. 45 shows analysis of the Z9/Z11-16OH titers of HaFAR mutant library upon expression in Y. lipolytica SPV603. * Indicates a second copy of the HaFAR enzyme in addition to the existing copy of the parental strain.

FIG. 46 shows analysis of the Z9/Z11-16Acid titers of HaFAR mutant library upon expression in Y. lipolytica SPV603. * Indicates a second copy of the HaFAR enzyme in addition to the existing copy in the parental strain.

FIG. 47 shows analysis of the fatty alcohol titers of selected strains expressing HaFAR and derived mutants. Strains were cultivated in shake flasks over a period of 72 h after addition of 10 g/L methyl palmitate. * Indicates a second copy of the HaFAR enzyme in addition to the existing copy in the parental strain. The analysis is based on technical quadruplicates.

FIG. 48 shows analysis of the fatty acid titers of selected strains expressing HaFAR and derived mutants. Strains were cultivated in shake flasks over a period of 72 h after addition of 10 g/L methyl palmitate. * Indicates a second copy of the HaFAR enzyme in addition to the existing copy in the parental strain. The analysis is based on technical quadruplicates.

FIG. 49 shows analysis of the fatty alcohol titers of selected strains expressing HaFAR and derived mutants. Strains were cultivated in shake flasks over a period of 20 h upon addition of 10 g/L methyl palmitate. The analysis is based on technical quadruplicates.

FIG. 50 shows analysis of the fatty alcohol titers of selected strains in a time course experiment in shake flasks. A copy of the enzyme HaFAR or HaS195A was introduced into the strains SPV1053 (Δdga1 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) and SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Cultivation was performed as biological triplicates in shake flasks. Strains were cultivated in shake flasks over a period of 72 h upon addition of 10 g/L methyl palmitate.

FIG. 51 shows analysis of the fatty acid titers of selected strains in a time course experiment in shake flasks. A copy of the enzyme HaFAR or HaS195A was introduced into the strains SPV1053 (Δdga1 ΔURA, ΔLeu, leu2::pTEF-HZ_Z1_desat_Hs-tXPR2_loxP) and SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Cultivation was performed as biological triplicates in shake flasks. Strains were cultivated in shake flasks over a period of 72 h upon addition of 10 g/L methyl palmitate.

FIG. 52 shows analysis of the fatty alcohol titers of new strains in a FAR library screening in 24 well plates. A copy of each respective FAR enzyme from Table 24 was introduced into the strain SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Cultivation was performed as biological quadruplicates in 24 well plates. Strains were cultivated over a period of 96 h upon addition of 10 g/L methyl palmitate.

FIG. 53 shows analysis of the fatty acid titers of new strains in a FAR library screening in 24 well plates. A single copy of each respective FAR enzyme from Table 24 was introduced into the strain SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Cultivation was performed as biological quadruplicates in 24 well plates. Strains were cultivated over a period of 96 h upon addition of 10 g/L methyl palmitate.

FIG. 54 shows a biosynthetic pathway capable of using tetradecyl-ACP (14:ACP) inputs to produce a blend of E- and Z-tetradecenyl acetate (E11-14:OAc and Z11-14:OAC) pheromones in a recombinant microorganism of the present disclosure.

SEQUENCES

A sequence listing for SEQ ID NO: 1-SEQ ID NO: 105 is part of this application and is incorporated by reference herein. The sequence listing is provided at the end of this document, and is separately provided in computer readable format.

DETAILED DESCRIPTION
Definitions

The following definitions and abbreviations are to be used for the interpretation of the disclosure.

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pheromone” includes a plurality of such pheromones and reference to “the microorganism” includes reference to one or more microorganisms, and so forth.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having, “contains,” “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or.”

The terms “about” and “around,” as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If “X” were the value, “about X” or “around X” would indicate a value from 0.9X to 1.1X, or, in some embodiments, a value from 0.95X to 1.05X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”

As used herein, the terms “microbial,” “microbial organism,” and “microorganism” include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a chemical.

As described herein, in some embodiments, the recombinant microorganisms are prokaryotic microorganism. In some embodiments, the prokaryotic microorganisms are bacteria. “Bacteria”, or “eubacteria”, refers to a domain of prokaryotic organisms. Bacteria include at least eleven distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic+non-photosynthetic Gram-negative bacteria (includes most “common” Gram-negative bacteria): (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces: (6) Bacteroides, Flavobacteria: (7) Chlamydia; (8) Green sulfur bacteria: (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibro, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, and sporulating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

The term “recombinant microorganism” and “recombinant host cell” are used interchangeably herein and refer to microorganisms that have been genetically modified to express or to overexpress endogenous enzymes, to express heterologous enzymes, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene. By “alteration” it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration. For example, the term “alter” can mean “inhibit,” but the use of the word “alter” is not limited to this definition. It is understood that the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The term “expression” with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein results from transcription and translation of the open reading frame sequence. The level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et al., 1989, supra.

The term “polynucleotide” is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids. The term “nucleoside” refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term “nucleotide analog” or “nucleoside analog” refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide.

It is understood that the polynucleotides described herein include “genes” and that the nucleic acid molecules described herein include “vectors” or “plasmids.” Accordingly, the term “gene”, also called a “structural gene” refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions, including introns, 5′-untranslated region (UTR), and 3′-UTR, as well as the coding sequence.

The term “enzyme” as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide or polypeptides, but can include enzymes composed of a different molecule including polynucleotides.

As used herein, the term “non-naturally occurring,” when used in reference to a microorganism organism or enzyme activity of the disclosure, is intended to mean that the microorganism organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microorganism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous, or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary non-naturally occurring microorganism or enzyme activity includes the hydroxylation activity described above.

The term “exogenous” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

On the other hand, the term “endogenous” or “native” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

The term “an endogenous or exogenous source of saturated C₆-C₂₄fatty acid” as used herein refers to a source of saturated C₆-C₂₄fatty acid originating from within the microorganism (endogenous), such as when a saturated C₆-C₂₄fatty acid is produced or synthesized inside the microorganism, or originating from outside the microorganism (exogenous), such as when a saturated C₆-C₂₄fatty acid is provided to the microorganism during the course of culturing or cultivating the microorganism in media in flasks or other containers.

The term “heterologous” as used herein in the context of a modified host cell refers to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true; (a) the molecule(s) is/are foreign (“exogenous”) to (i.e., not naturally found in) the host cell; (b) the molecule(s) is/are naturally found in (e.g., is “endogenous to”) a given host microorganism or host cell but is either produced in an unnatural location or in an unnatural amount in the cell, and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s) such that the molecule differing in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid as found endogenously is produced in an unnatural (e.g., greater than naturally found) amount in the cell.

As used herein, the term “homologous sequences” “homolog” “homologs” or “orthologs” refers to related sequences (nucleic or amino acid) that are functionally related to the referenced sequence. A functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and/or (b) the same or similar biological function. Use of the term homolog in this disclosure refers to instances in which both (a) and (b) are indicated. The degree of sequence identity may vary, but in one embodiment, is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity when using standard sequence alignment programs known in the art (e.g., Clustal Omega alignment using default parameters). Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus (Scientific and Educational Software, Pennsylvania). Other non-limiting alignment programs include Sequencher (Gene Codes, Ann Arbor, Mich.), AlignX, and Vector NTI (Invitrogen, Carlsbad, Calif.). Thus, a reference to a homolog in the present disclosure will be understood as referencing a related sequence with the same or similar biological function, and a high degree of sequence identity as described above.

The term “fatty acid” as used herein refers to a compound of structure R—COOH, wherein R is a C₆to C₂₄saturated, unsaturated, linear, branched or cyclic hydrocarbon and the carboxyl group is at position 1. In a particular embodiment. R is a C₆to C₂₄saturated or unsaturated linear hydrocarbon and the carboxyl group is at position 1.

The term “fatty alcohol” as used herein refers to an aliphatic alcohol having the formula R—OH, wherein R is a C₆to C₂₄saturated, unsaturated, linear, branched or cyclic hydrocarbon. In a particular embodiment, R is a C₆to C₂₄saturated or unsaturated linear hydrocarbon.

The term “fatty acyl-CoA” refers to a compound having the structure R—(CO)—S—R₁, wherein R₁is Coenzyme A, and the term “fatty acyl-ACP” refers to a compound having the structure R—(CO)—S—R₁, wherein R₁is an acyl carrier protein ACP.

The term “short chain” or “short-chain” refers to fatty alcohols, fatty aldehydes, and/or fatty acetates, including pheromones, fragrances, flavors, and polymer intermediates with carbon chain length shorter than or equal to C18.

Introduction

The present disclosure addresses the need for novel technologies for the cost-efficient production of valuable products from low-cost feedstocks. Specifically, the present inventors have addressed this need with the development of recombinant microorganisms capable of producing a wide-range of unsaturated C₆-C₂₄fatty alcohols, aldehydes, and acetates including synthetic insect pheromones, fragrances, flavors, and polymer intermediates from low-cost feedstocks. Thus, aspects of the disclosure are based on the inventors' discovery that recombinant microorganisms can be engineered in order to produce valuable products from low-cost feedstocks, which circumvents conventional synthetic methodologies to produce valuable products.

As discussed above, recombinant microorganisms can be engineered to synthesize mono- or poly-unsaturated C₆-C₂₄fatty alcohols. Mono- or poly-unsaturated C₆-C₂₄fatty alcohols synthesized as described herein can be further converted into the corresponding aldehydes or acetates. Thus, various embodiments of the present disclosure can be used to synthesize a variety of insect pheromones selected from fatty alcohols, aldehydes, and acetates. Additionally, embodiments described herein can also be used for the synthesis of fragrances, flavors, and polymer intermediates.

Engineering of the microbial hosts entail the expression of a non-native pheromone biosynthetic pathway which is comprised of but not limited to one or multiple fatty acyl desaturases, and fatty alcohol-forming or fatty aldehyde-forming reductases. Fatty acids produced by desaturation reactions can be stored intracellularly as triacylglycerides or reduced enzymatically by reductases to form fatty alcohols or aldehydes. Triacylglycerides containing unsaturated fatty acids can be extracted, esterified, and chemically reduced to produce unsaturated fatty alcohols. Fatty alcohols produced via the described pathways can be further converted into fatty aldehyde pheromones, and fatty acetate pheromones via subsequent chemical oxidation, and esterification methods, respectively. Methods of chemical oxidation and esterification are known in the arts. Fatty alcohols produced via the described pheromone biosynthetic pathway can also be further converted into fatty aldehyde pheromones, and fatty acetate pheromones using enzymatic conversion such as alcohol dehydrogenases, and acetyltransferase, respectively. Similarly, fatty acyl-CoA or fatty acyl-ACP formed as intermediates in the pheromone biosynthetic pathway can be released as free fatty acids by native or heterologously derived thioesterases, to become substrates for synthesis of pheromones using metathesis.

Pheromones

As described above, embodiments of the disclosure provide for the synthesis of one or more insect pheromones using a recombinant microorganism. A pheromone is a volatile chemical compound that is secreted by a particular insect for the function of chemical communication within the species. That is, a pheromone is secreted or excreted chemical factor that triggers a social response in members of the same species. There are, inter alia, alarm pheromones, food trail pheromones, sex pheromones, aggregation pheromones, epideictic pheromones, releaser pheromones, primer pheromones, and territorial pheromones, that affect behavior or physiology.

Non-limiting examples of insect pheromones which can be synthesized using the recombinant microorganisms and methods disclosed herein include linear alcohols, aldehydes, and acetates listed in Table 1.

TABLE 1

C₆-C₂₀Linear Pheromones

Name
Name

(E)-2-Decen-1-ol
(E,E)-10,12-Tetradecadien-1-ol

(E)-2-Decenyl acetate
(E,E)-10,12-Tetradecadienyl acetate

(E)-2-Decenal
(E,E)-10,12-Tetradecadienal

(Z)-2-Decen-1-ol
(E,Z)-10,12-Tetradecadienyl acetate

(Z)-2-Decenyl acetate
(Z,E)-10,12-Tetradecadienyl acetate

(Z)-2-Decenal
(Z,Z)-10,12-Tetradecadien-1-ol

(E)-3-Decen-1-ol
(Z,Z)-10,12-Tetradecadienyl acetate

(Z)-3-Decenyl acetate
(E,Z,Z)-3,8,11-Tetradecatrienyl

acetate

(Z)-3-Decen-1-ol
(E)-8-Pentadecen-1-ol

(Z)-4-Decen-1-ol
(E)-8-Pentadecenyl acetate

(E)-4-Decenyl acetate
(Z)-8-Pentadecen-1-ol

(Z)-4-Decenyl acetate
(Z)-8-Pentadecenyl acetate

(Z)-4-Decenal
(Z)-9-Pentadecenyl acetate

(E)-5-Decen-1-ol
(E)-9-Pentadecenyl acetate

(E)-5-Decenyl acetate
(Z)-10-Pentadecenyl acetate

(Z)-5-Decen-1-ol
(Z)-10-Pentadecenal

(Z)-5-Decenyl acetate
(E)-12-Pentadecenyl acetate

(Z)-5-Decenal
(Z)-12-Pentadecenyl acetate

(E)-7-Decenyl acetate
(Z,Z)-6,9-Pentadecadien-1-ol

(Z)-7-Decenyl acetate
(Z,Z)-6,9-Pentadecadienyl acetate

(E)-8-Decen-1-ol
(Z,Z)-6,9-Pentadecadienal

(E,E)-2,4-Decadienal
(E,E)-8,10-Pentadecadienyl acetate

(E,Z)-2,4-Decadienal
(E,Z)-8,10-Pentadecadien-1-ol

(Z,Z)-2,4-Decadienal
(E,Z)-8,10-Pentadecadienyl acetate

(E,E)-3,5-Decadienyl acetate
(Z,E)-8,10-Pentadecadienyl acetate

(Z,E)-3,5-Decadienyl acetate
(Z,Z)-8,10-Pentadecadienyl acetate

(Z,Z)-4,7-Decadien-1-ol
(E,Z)-9,11-Pentadecadienal

(Z,Z)-4,7-Decadienyl acetate
(Z,Z)-9,11-Pentadecadienal

(E)-2-Undecenyl acetate
(Z)-3-Hexadecenyl acetate

(E)-2-Undecenal
(E)-5-Hexadecen-1-ol

(Z)-5-Undecenyl acetate
(E)-5-Hexadecenyl acetate

(Z)-7-Undecenyl acetate
(Z)-5-Hexadecen-1-ol

(Z)-8-Undecenyl acetate
(Z)-5-Hexadecenyl acetate

(Z)-9-Undecenyl acetate
(E)-6-Hexadecenyl acetate

(E)-2-Dodecenal
(E)-7-Hexadecen-1-ol

(Z)-3-Dodecen-1-ol
(E)-7-Hexadecenyl acetate

(E)-3-Dodecenyl acetate
(E)-7-Hexadecenal

(Z)-3-Dodecenyl acetate
(Z)-7-Hexadecen-1-ol

(E)-4-Dodecenyl acetate
(Z)-7-Hexadecenyl acetate

(E)-5-Dodecen-1-ol
(Z)-7-Hexadecenal

(E)-5-Dodecenyl acetate
(E)-8-Hexadecenyl acetate

(Z)-5-Dodecen-1-ol
(E)-9-Hexadecen-1-ol

(Z)-5-Dodecenyl acetate
(E)-9-Hexadecenyl acetate

(Z)-5-Dodecenal
(E)-9-Hexadecenal

(E)-6-Dodecen-1-ol
(Z)-9-Hexadecen-1-ol

(Z)-6-Dodecenyl acetate
(Z)-9-Hexadecenyl acetate

(E)-6-Dodecenal
(Z)-9-Hexadecenal

(E)-7-Dodecen-1-ol
(E)-10-Hexadecen-1-ol

(E)-7-Dodecenyl acetate
(E)-10-Hexadecenal

(E)-7-Dodecenal
(Z)-10-Hexadecenyl acetate

(Z)-7-Dodecen-1-ol
(Z)-10-Hexadecenal

(Z)-7-Dodecenyl acetate
(E)-11-Hexadecen-1-ol

(Z)-7-Dodecenal
(E)-11-Hexadecenyl acetate

(E)-8-Dodecen-1-ol
(E)-11-Hexadecenal

(E)-8-Dodecenyl acetate
(Z)-11-Hexadecen-1-ol

(E)-8-Dodecenal
(Z)-11-Hexadecenyl acetate

(Z)-8-Dodecen-1-ol
(Z)-11-Hexadecenal

(Z)-8-Dodecenyl acetate
(Z)-12-Hexadecenyl acetate

(E)-9-Dodecen-1-ol
(Z)-12-Hexadecenal

(E)-9-Dodecenyl acetate
(E)-14-Hexadecenal

(E)-9-Dodecenal
(Z)-14-Hexadecenyl acetate

(Z)-9-Dodecen-1-ol
(E,E)-1,3-Hexadecadien-1-ol

(Z)-9-Dodecenyl acetate
(E,Z)-4,6-Hexadecadien-1-ol

(Z)-9-Dodecenal
(E,Z)-4,6-Hexadecadienyl acetate

(E)-10-Dodecen-1-ol
(E,Z)-4,6-Hexadecadienal

(E)-10-Dodecenyl acetate
(E,Z)-6,11-Hexadecadienyl acetate

(E)-10-Dodecenal
(E,Z)-6,11-Hexadecadienal

(Z)-10-Dodecen-1-ol
(Z,Z)-7,10-Hexadecadien-1-ol

(Z)-10-Dodecenyl acetate
(Z,Z)-7,10-Hexadecadienyl acetate

(E,Z)-3,5-Dodecadienyl acetate
(Z,E)-7,11-Hexadecadien-1-ol

(Z,E)-3,5-Dodecadienyl acetate
(Z,E)-7,11-Hexadecadienyl acetate

(Z,Z)-3,6-Dodecadien-1-ol
(Z,E)-7,11-Hexadecadienal

(E,E)-4,10-Dodecadienyl acetate
(Z,Z)-7,11-Hexadecadien-1-ol

(E,E)-5,7-Dodecadien-1-ol
(Z,Z)-7,11-Hexadecadienyl acetate

(E,E)-5,7-Dodecadienyl acetate
(Z,Z)-7,11-Hexadecadienal

(E,Z)-5,7-Dodecadien-1-ol
(Z,Z)-8,10-Hexadecadienyl acetate

(E,Z)-5,7-Dodecadienyl acetate
(E,Z)-8,11-Hexadecadienal

(E,Z)-5,7-Dodecadienal
(E,E)-9,11-Hexadecadienal

(Z,E)-5,7-Dodecadien-1-ol
(E,Z)-9,11-Hexadecadienyl acetate

(Z,E)-5,7-Dodecadienyl acetate
(E,Z)-9,11-Hexadecadienal

(Z,E)-5,7-Dodecadienal
(Z,E)-9,11-Hexadecadienal

(Z,Z)-5,7-Dodecadienyl acetate
(Z,Z)-9,11-Hexadecadienal

(Z,Z)-5,7-Dodecadienal
(E,E)-10,12-Hexadecadien-1-ol

(E,E)-7,9-Dodecadienyl acetate
(E,E)-10,12-Hexadecadienyl acetate

(E,Z)-7,9-Dodecadien-1-ol
(E,E)-10,12-Hexadecadienal

(E,Z)-7,9-Dodecadienyl acetate
(E,Z)-10,12-Hexadecadien-1-ol

(E,Z)-7,9-Dodecadienal
(E,Z)-10,12-Hexadecadienyl acetate

(Z,E)-7,9-Dodecadien-1-ol
(E,Z)-10,12-Hexadecadienal

(Z,E)-7,9-Dodecadienyl acetate
(Z,E)-10,12-Hexadecadienyl acetate

(Z,Z)-7,9-Dodecadien-1-ol
(Z,E)-10,12-Hexadecadienal

(Z,Z)-7,9-Dodecadienyl acetate
(Z,Z)-10,12-Hexadecadienal

(E,E)-8,10-Dodecadien-1-ol
(E,E)-11,13-Hexadecadien-1-ol

(E,E)-8,10-Dodecadienyl acetate
(E,E)-11,13-Hexadecadienyl acetate

(E,E)-8,10-Dodecadienal
(E,E)-11,13-Hexadecadienal

(E,Z)-8,10-Dodecadien-1-ol
(E,Z)-11,13-Hexadecadien-1-ol

(E,Z)-8,10-Dodecadienyl acetate
(E,Z)-11,13-Hexadecadienyl acetate

(E,Z)-8,10-Dodecadienal
(E,Z)-11,13-Hexadecadienal

(Z,E)-8,10-Dodecadien-1-ol
(Z,E)-11,13-Hexadecadien-1-ol

(Z,E)-8,10-Dodecadienyl acetate
(Z,E)-11,13-Hexadecadienyl acetate

(Z,E)-8,10-Dodecadienal
(Z,E)-11,13-Hexadecadienal

(Z,Z)-8,10-Dodecadien-1-ol
(Z,Z)-11,13-Hexadecadien-1-ol

(Z,Z)-8,10-Dodecadienyl acetate
(Z,Z)-11,13-Hexadecadienyl acetate

(Z,E,E)-3,6,8-Dodecatrien-1-ol
(Z,Z)-11,13-Hexadecadienal

(Z,Z,E)-3,6,8-Dodecatrien-1-ol
(E,E)-10,14-Hexadecadienal

(E)-2-Tridecenyl acetate
(Z,E)-11,14-Hexadecadienyl acetate

(Z)-2-Tridecenyl acetate
(E,E,Z)-4,6,10-Hexadecatrien-1-ol

(E)-3-Tridecenyl acetate
(E,E,Z)-4,6,10-Hexadecatrienyl

acetate

(E)-4-Tridecenyl acetate
(E,Z,Z)-4,6,10-Hexadecatrien-1-ol

(Z)-4-Tridecenyl acetate
(E,Z,Z)-4,6,10-Hexadecatrienyl

acetate

(Z)-4-Tridecenal
(E,E,Z)-4,6,11-Hexadecatrienyl

acetate

(E)-6-Tridecenyl acetate
(E,E,Z)-4,6,11-Hexadecatrienal

(Z)-7-Tridecenyl acetate
(Z,Z,E)-7,11,13-Hexadecatrienal

(E)-8-Tridecenyl acetate
(E,E,E)-10,12,14-Hexadecatrienyl

acetate

(Z)-8-Tridecenyl acetate
(E,E,E)-10,12,14-Hexadecatrienal

(E)-9-Tridecenyl acetate
(E,E,Z)-10,12,14-Hexadecatrienyl

acetate

(Z)-9-Tridecenyl acetate
(E,E,Z)-10,12,14-Hexadecatrienal

(Z)-10-Tridecenyl acetate
(E,E,Z,Z)-4,6,11,13-

Hexadecatetraenal

(E)-11-Tridecenyl acetate
(E)-2-Heptadecenal

(Z)-11-Tridecenyl acetate
(Z)-2-Heptadecenal

(E,Z)-4,7-Tridecadienyl acetate
(E)-8-Heptadecen-1-ol

(Z,Z)-4,7-Tridecadien-1-ol
(E)-8-Heptadecenyl acetate

(Z,Z)-4,7-Tridecadienyl acetate
(Z)-8-Heptadecen-1-ol

(E,Z)-5,9-Tridecadienyl acetate
(Z)-9-Heptadecenal

(Z,E)-5,9-Tridecadienyl acetate
(E)-10-Heptadecenyl acetate

(Z,Z)-5,9-Tridecadienyl acetate
(Z)-11-Heptadecen-1-ol

(Z,Z)-7,11-Tridecadienyl acetate
(Z)-11-Heptadecenyl acetate

(E,Z,Z)-4,7,10-Tridecatrienyl
(E,E)-4,8-Heptadecadienyl acetate

acetate

(E)-3-Tetradecen-1-ol
(Z,Z)-8,10-Heptadecadien-1-ol

(E)-3-Tetradecenyl acetate
(Z,Z)-8,11-Heptadecadienyl acetate

(Z)-3-Tetradecen-1-ol
(E)-2-Octadecenyl acetate

(Z)-3-Tetradecenyl acetate
(E)-2-Octadecenal

(E)-5-Tetradecen-1-ol
(Z)-2-Octadecenyl acetate

(E)-5-Tetradecenyl acetate
(Z)-2-Octadecenal

(E)-5-Tetradecenal
(E)-9-Octadecen-1-ol

(Z)-5-Tetradecen-1-ol
(E)-9-Octadecenyl acetate

(Z)-5-Tetradecenyl acetate
(E)-9-Octadecenal

(Z)-5-Tetradecenal
(Z)-9-Octadecen-1-ol

(E)-6-Tetradecenyl acetate
(Z)-9-Octadecenyl acetate

(Z)-6-Tetradecenyl acetate
(Z)-9-Octadecenal

(E)-7-Tetradecen-1-ol
(E)-11-Octadecen-1-ol

(E)-7-Tetradecenyl acetate
(E)-11-Octadecenal

(Z)-7-Tetradecen-1-ol
(Z)-11-Octadecen-1-ol

(Z)-7-Tetradecenyl acetate
(Z)-11-Octadecenyl acetate

(Z)-7-Tetradecenal
(Z)-11-Octadecenal

(E)-8-Tetradecenyl acetate
(E)-13-Octadecenyl acetate

(Z)-8-Tetradecen-1-ol
(E)-13-Octadecenal

(Z)-8-Tetradecenyl acetate
(Z)-13-Octadecen-1-ol

(Z)-8-Tetradecenal
(Z)-13-Octadecenyl acetate

(E)-9-Tetradecen-1-ol
(Z)-13-Octadecenal

(E)-9-Tetradecenyl acetate
(E)-14-Octadecenal

(Z)-9-Tetradecen-1-ol
(E,Z)-2,13-Octadecadien-1-ol

(Z)-9-Tetradecenyl acetate
(E,Z)-2,13-Octadecadienyl acetate

(Z)-9-Tetradecenal
(E,Z)-2,13-Octadecadienal

(E)-10-Tetradecenyl acetate
(Z,E)-2,13-Octadecadienyl acetate

(Z)-10-Tetradecenyl acetate
(Z,Z)-2,13-Octadecadien-1-ol

(E)-11-Tetradecen-1-ol
(Z,Z)-2,13-Octadecadienyl acetate

(E)-11-Tetradecenyl acetate
(E,E)-3,13-Octadecadienyl acetate

(E)-11-Tetradecenal
(E,Z)-3,13-Octadecadienyl acetate

(Z)-11-Tetradecen-1-ol
(E,Z)-3,13-Octadecadienal

(Z)-11-Tetradecenyl acetate
(Z,E)-3,13-Octadecadienyl acetate

(Z)-11-Tetradecenal
(Z,Z)-3,13-Octadecadienyl acetate

(E)-12-Tetradecenyl acetate
(Z,Z)-3,13-Octadecadienal

(Z)-12-Tetradecenyl acetate
(E,E)-5,9-Octadecadien-1-ol

(E,E)-2,4-Tetradecadienal
(E,E)-5,9-Octadecadienyl acetate

(E,E)-3,5-Tetradecadienyl acetate
(E,E)-9,12-Octadecadien-1-ol

(E,Z)-3,5-Tetradecadienyl acetate
(Z,Z)-9,12-Octadecadienyl acetate

(Z,E)-3,5-Tetradecadienyl acetate
(Z,Z)-9,12-Octadecadienal

(E,Z)-3,7-Tetradecadienyl acetate
(Z,Z)-11,13-Octadecadienal

(E,Z)-3,8-Tetradecadienyl acetate
(E,E)-11,14-Octadecadienal

(E,Z)-4,9-Tetradecadienyl acetate
(Z,Z)-13,15-Octadecadienal

(E,Z)-4,9-Tetradecadienal
(Z,Z,Z)-3,6,9-Octadecatrienyl

acetate

(E,Z)-4,10-Tetradecadienyl acetate
(E,E,E)-9,12,15-Octadecatrien-1-ol

(E,E)-5,8-Tetradecadienal
(Z,Z,Z)-9,12,15-Octadecatrienyl

acetate

(Z,Z)-5,8-Tetradecadien-1-ol
(Z,Z,Z)-9,12,15-Octadecatrienal

(Z,Z)-5,8-Tetradecadienyl acetate

(Z,Z)-5,8-Tetradecadienal

(E,E)-8,10-Tetradecadien-1-ol

(E,E)-8,10-Tetradecadienyl acetate

(E,E)-8,10-Tetradecadienal

(E,Z)-8,10-Tetradecadienyl acetate

(E,Z)-8,10-Tetradecadienal

(Z,E)-8,10-Tetradecadien-1-ol

(Z,E)-8,10-Tetradecadienyl acetate

(Z,Z)-8,10-Tetradecadienal

(E,E)-9,11-Tetradecadienyl acetate

(E,Z)-9,11-Tetradecadienyl acetate

(Z,E)-9,11-Tetradecadien-1-ol

(Z,E)-9,11-Tetradecadienyl acetate

(Z,E)-9,11-Tetradecadienal

(Z,Z)-9,11-Tetradecadien-1-ol

(Z,Z)-9,11-Tetradecadienyl acetate

(Z,Z)-9,11-Tetradecadienal

(E,E)-9,12-Tetradecadienyl acetate

(Z,E)-9,12-Tetradecadien-1-ol

(Z,E)-9,12-Tetradecadienyl acetate

(Z,E)-9,12 -Tetradecadienal

(Z,Z)-9,12-Tetradecadien-1-ol

(Z,Z)-9,12-Tetradecadienyl acetate

In some aspects, the pheromones synthesized as taught in this disclosure include at least one pheromone listed in Table 2a to modulate the behavior of an insect listed in Table 2a. In other aspects, non-limiting examples of insect pheromones which can be synthesized using the recombinant microorganisms and methods disclosed herein include alcohols, aldehydes, and acetates listed in Table 2a. However, the microorganisms described herein are not limited to the synthesis of C₆-C₂₀pheromones listed in Table 1 and Table 2a. Rather, the disclosed microorganisms can also be utilized in the synthesis of various C₆-C₂₄mono- or poly-unsaturated fatty alcohols, aldehydes, and acetates, including fragrances, flavors, and polymer intermediates.

TABLE 2a

Exemplary pheromones that can be synthesized according to methods described in

the present disclosure

Example of Biological

Name
Structure
importance

(Z)-3-hexen-1-ol

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See, Sugimoto et al. (2014)

(Z)-3-nonen-1-ol

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West Indian Fruity Fly male sex pheromone

(Z)-5-decen-1-ol

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(Z)-5-decenyl acetate

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Agrotis segetum sex pheromone component

(E)-5-decen-1-ol

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Anarsia lineatella sex pheromone component

(E)-5-decenyl acetate

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Anarsia lineatella sex pheromone component

(Z)-7-dodecen-1-ol

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(Z)-7-dodecenyl acetate

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Pseudoplusia includens sex pheromone

Argrotis segetum sex pheromone

component

(E)-8-dodcen-1-ol

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Citrus Fruit Moth sex pheromone

(E)-8-dodecenyl acetate

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Grapholitha molesta, Ecdytolopha aurantiana sex

pheromone component

(Z)-8-dodecen-1-ol

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Grapholitha molesta, Ecdytolopha aurantiana sex pheromone component

(Z)-8-dodecenyl acetate

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Grapholitha molesta sex pheromone component

(Z)-9-dodecen-1-ol

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(Z)-9-dodecenyl acetate

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Eupoecilia ambiguella sex pheromone

(E,E)-8,10-dodecadien- 1-ol

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Cydia pomonella

(7E,9Z)-dodecadienyl acetate

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Lobesia botrana

(Z)-9-tetradecen-1-ol

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(Z)-9-tetradecenyl acetate

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Pandemis pyrusana, Narangaaenescens, Agrotis segetum sex

pheromone component

(Z)-11-tetradecen-1-ol

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(Z)-11-tetradecenyl acetate

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Pandemis pyrusana, Choristoneura roseceana sex

pheromone component

(E)-11-tetradecen-1-ol

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(E)-11-tetradecenyl acetate

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Choristoneura roseceana, Crocidolomia pavonana sex pheromone component

(Z)-7-hexadecen-1-ol

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(Z)-7-hexadecenal

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Diatraea considerata sex pheromone component

(Z)-9-hexadecen-1-ol

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(Z)-9-hexadecenal

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Helicoverpa zea, Helicoverpaamigera, Heliothis virescens sex

pheromone component

(Z)-9-hexadecenyl acetate

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Naranga aenescens sex pheromone component

(Z)-11-hexadecen-1-ol

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(Z)-11-hexadecenal

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Platyptila carduidactyla, Heliothisvirescens sex pheromone Helicoverpa zea, Helicoverpa

armigera, Plutella xylostella,

Diatraea considerate, Diatraea

grandiosella, Diatraea

saccharalis, Acrolepiopsis

assectella sex pheromone

component

(Z)-11-hexadecenyl acetate

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Discestra trifolii sex pheromone Heliothis virescens, Plutella

xylostella, Acrolespiopsis

assectella, Crocidolomia

pavonana, Naranga aenescens

sex pheromone component

(Z,Z)-11,13- hexadecadienal

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Amyelosis transitella

(Z)-11,13- hexadecadian-1-ol

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Amyelosis transitella

(11Z,13E)- hexadecadien-1-ol

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Amyelosis transitella

(9Z,11E)- hexadecadienal

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(Z)-13-octadecen-1-ol

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(Z)-13-octadecenal

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Diatraea considerata, Diatraeagrandiosella sex pheromone

component

(Z,Z,Z,Z,Z)- 3,6,9,12,15- tricosapentaene

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Amyelosis transitella

Most pheromones comprise a hydrocarbon skeleton with the terminal hydrogen substituted by a functional group (Ryan M F (2002). Insect Chemoreception. Fundamental and Applied. Kluwer Academic Publishers). Table 2b shows some common functional groups, along with their formulas, prefixes and suffixes. The presence of one or more double bonds, generated by the loss of hydrogens from adjacent carbons, determines the degree of unsaturation of the molecule and alters the designation of a hydrocarbon from -ane (no multiple bonds) to -ene. The presence of two and three double bonds is indicated by ending the name with -diene and -triene, respectively. The position of each double bond is represented by a numeral corresponding to that of the carbon from which it begins, with each carbon numbered from that attached to the functional group. The carbon to which the functional group is attached is designated -1-. Pheromones may have, but are not limited to, hydrocarbon chain lengths numbering 10 (deca-), 12 (dodeca-), 14 (tetradeca-), 16 (hexadeca-), or 18 (octadeca-) carbons long. The presence of a double bond has another effect. It precludes rotation of the molecule by fixing it in one of two possible configurations, each representing geometric isomers that are different molecules. These are designated either E (from the German word Entgegen, opposite) or Z (Zusammen, together), when the carbon chains are connected on the opposite (trans) or same (cis) side, respectively, of the double bond.

TABLE 2b

Prefixes and suffixes for common functional groups

Functional group
Formula
Prefix
Suffix

Alcohol
—OH
Hydroxy-
-ol

Aldehyde
—CH═O
Formyl-
-al

Amine
—NH₂
Amino-
-amine

Carboxylic add
—COOH
Carboxy-
-oic add

Ester
—COOR
R-oxycarbonyl
-R-oate

Ketone
>C═O
Oxo-
-one

From Howse, P E, Stevens, I D R and Jones, O T (1998). Insect pheromones and their use in pest management. London: Chapman and Hall.

Pheromones described herein can be referred to using IUPAC nomenclature or various abbreviations or variations known to one skilled in the art. For example, (11Z)-hexadecen-1-al, can also be written as Z-11-hexadecen-1-al, Z-11-hexadecenal, or Z-x-y:Ald, wherein x represents the position of the double bond and y represents the number of carbons in the hydrocarbon skeleton. Abbreviations used herein and known to those skilled in the art to identify functional groups on the hydrocarbon skeleton include “Ald,” indicating an aldehyde. “OH,” indicating an alcohol, and “Ac,” indicating an acetyl. Also, the number of carbons in the chain can be indicated using numerals rather than using the written name. Thus, as used herein, an unsaturated carbon chain comprised of sixteen carbons can be written as hexadecene or 16.

Similar abbreviation and derivations are used herein to describe pheromone precursors. For example, the fatty acyl-CoA precursors of (11Z)-hexadecen-1-al can be identified as (11Z)-hexadecenyl-CoA or Z-11-16:Acyl-CoA.

The present disclosure relates to the synthesis of mono- or poly-unsaturated C₆-C₂₄fatty alcohols, aldehydes, and acetates using a recombinant microorganism comprised of one or more heterologous enzymes, which catalyze substrate to product conversions for one or more steps in the synthesis process.

Desaturase

The present disclosure describes enzymes that desaturate fatty acyl substrates to corresponding unsaturated fatty acyl substrates.

In some embodiments, a desaturase is used to catalyze the conversion of a fatty acyl-CoA or acyl-ACP to a corresponding unsaturated fatty acyl-CoA or acyl-ACP. A desaturase is an enzyme that catalyzes the formation of a carbon-carbon double bond in a saturated fatty acid or fatty acid derivative, e.g., fatty acyl-CoA or fatty acyl-ACP (collectively referred to herein as “fatty acyl”), by removing at least two hydrogen atoms to produce a corresponding unsaturated fatty acid/acyl. Desaturases are classified with respect to the ability of the enzyme to selectively catalyze double bond formation at a subterminal carbon relative to the methyl end of the fatty acid/acyl or a subterminal carbon relative to the carbonyl end of the fatty acid/acyl. Omega (w) desaturases catalyze the formation of a carbon-carbon double bond at a fixed subterminal carbon relative to the methyl end of a fatty acid/acyl. For example, an ω³desaturase catalyzes the formation of a double bond between the third and fourth carbon relative the methyl end of a fatty acid/acyl. Delta (Δ) desaturases catalyze the formation of a carbon-carbon double bond at a specific position relative to the carboxyl group of a fatty acid or the carbonyl group of a fatty acyl CoA. For example, a Δ⁹desaturase catalyzes the formation of a double bond between the C₉and C₁₀carbons with respect to the carboxyl end of the fatty acid or the carbonyl group of a fatty acyl CoA.

As used herein, a desaturase can be described with reference to the location in which the desaturase catalyzes the formation of a double bond and the resultant geometric configuration (i.e., E/Z) of the unsaturated hydrocarbon. Accordingly, as used herein, a Z9 desaturase refers to a Δ desaturase that catalyzes the formation of a double bond between the C₉and C₁₀carbons with respect to the carbonyl end of a fatty acid/acyl, thereby orienting two hydrocarbons on opposing sides of the carbon-carbon double bonds in the cis or Z configuration. Similarly, as used herein, a Z11 desaturase refers to a Δ desaturase that catalyzes the formation of a double bond between the C₁₁and C₁₂carbons with respect to the carbonyl end of a fatty acid/acyl.

Desaturases have a conserved structural motif. This sequence motif of transmembrane desaturases is characterized by [HX3-4HX7-41(3 non-His)HX2-3(1 nonHis)HHX61-189(40 non-His)HX2-3(1 non-His)HH]. The sequence motif of soluble desaturases is characterized by two occurrences of [D/EEXXH].

In some embodiments, the desaturase is a fatty acyl-CoA desaturase that catalyzes the formation of a double bond in a fatty acyl-CoA. In some such embodiments, the fatty acyl-CoA desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Thus, the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.

In some embodiments, the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal CoA on the unsaturated fatty acyl-CoA. Thus, in some embodiments, a desaturase can be selected for use in the recombinant microorganism which catalyzes double bond insertion at the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to the carbonyl group on a fatty acyl-CoA.

In some embodiments, the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond in a saturated fatty acyl-CoA such that the resultant unsaturated fatty acyl-CoA has a cis or trans (i.e., Z or E) geometric configuration.

In some embodiments, the desaturase is a fatty acyl-ACP desaturase that catalyzes the formation of a double bond in a fatty acyl-ACP. In some embodiments, the fatty acyl-ACP desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Thus, the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.

In some embodiments, the fatty acyl-ACP desaturase described herein is capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal carbonyl on the unsaturated fatty acyl-ACP. Thus, in some embodiments, a desaturase can be selected for use in the recombinant microorganism which catalyzes double bond insertion at the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to the carbonyl group on a fatty acyl-ACP.

In some embodiments, the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond in a saturated fatty acyl-CoA such that the resultant unsaturated fatty acyl-ACP has a cis or trans (i.e., Z or E) geometric configuration.

In one embodiment, the fatty acyl desaturase is a Z11 desaturase. In some embodiments, a nucleic acid sequence encoding a Z11 desaturase from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 9, 18, 24 and 26 from Trichoplusia ni. In some embodiments, the Z1 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 49 from Trichoplusia ni. In other embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 10 and 16 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 53 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 12, 17 and 30 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transitella. In further embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 13, 19, 25, 27 and 31 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 54 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 39 from S. inferens. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in GenBank Accession nos. AF416738, AGH12217.1, AH21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, a complete or partial Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of a Z11 desaturase is replaced by an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a nucleotide sequence selected from SEQ ID NOs: 15, 28 and 29. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 78, 79 and 80.

In one embodiment, the fatty acyl desaturase is a Z9 desaturase. In some embodiments, a nucleic acid sequence encoding a Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 20 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 58 from Ostrinia furnacalis. In other embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 60 from Helicoverpa zea.

Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a Z11 or Z9 desaturase exhibiting at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 39, 49, 50, 51, 52, 53, 54, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 78, 79, 80, 95, 97, 99, 101, 103, and 105.

Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding for a Z11 or Z9 desaturase, wherein said nucleic acid molecule exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 68, 69, 70, 71, 94, 96, 98, 100, 102, and 104.

In some embodiments, the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA.

Fatty Acyl Reductase

The present disclosure describes enzymes that reduce fatty acyl substrates to corresponding fatty alcohols or aldehydes.

In some embodiments, a fatty alcohol forming fatty acyl-reductase is used to catalyze the conversion of a fatty acyl-CoA to a corresponding fatty alcohol. In some embodiments, a fatty aldehyde forming fatty acyl-reductase is used to catalyze the conversion of a fatty acyl-ACP to a corresponding fatty aldehyde. A fatty acyl reductase is an enzyme that catalyzes the reduction of a fatty acyl-CoA to a corresponding fatty alcohol or the reduction of a fatty acyl-ACP to a corresponding fatty aldehyde. A fatty acyl-CoA and fatty acyl-ACP has a structure of R—(CO)—S—R₁, wherein R is a C₆to C₂₄saturated, unsaturated, linear, branched or cyclic hydrocarbon, and R₁represents CoA or ACP. In a particular embodiment, R is a C₆to C₂₄saturated or unsaturated linear hydrocarbon. “CoA” is a non-protein acyl carrier group involved in the synthesis and oxidation of fatty acids. “ACP” is an acyl carrier protein, i.e., a polypeptide or protein subunit, of fatty acid synthase used in the synthesis of fatty acids.

Thus, in some embodiments, the disclosure provides for a fatty alcohol forming fatty acyl-reductase which catalyzes the reduction of a fatty acyl-CoA to the corresponding fatty alcohol. For example, R—(CO)—S-CoA is converted to R—CH₂OH and CoA-SH when two molecules of NAD(P)H are oxidized to NAD(P)⁺. Accordingly, in some such embodiments, a recombinant microorganism described herein can include a heterologous fatty alcohol forming fatty acyl-reductase, which catalyzes the reduction a fatty acyl-CoA to the corresponding fatty alcohol. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase which catalyzes the conversion of a mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.

In other embodiments, the disclosure provides for a fatty aldehyde forming fatty acyl-reductase which catalyzes the reduction of a fatty acyl-ACP to the corresponding fatty aldehyde. For example, R—(CO)—S-ACP is converted to R—(CO)—H and ACP-SH when one molecule of NAD(P)H is oxidized to NAD(P)⁺. In some such embodiments, a recombinant microorganism described herein can include a heterologous fatty aldehyde forming fatty acyl-reductase, which catalyzes the reduction a fatty acyl-ACP to the corresponding fatty aldehyde. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acyl reductase which catalyzes the conversion of a mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty aldehyde.

In some insect species the respective alcohol-forming fatty acyl reductase (FAR) enzymes are activated via site specific dephosphorylation (Jurenka, R. & Rafaeli, A. Regulatory Role of PBAN in Sex Pheromone Biosynthesis of Heliothine Moths. Front. Endocrinol. (Lausanne), 2: 46 (2011); Gilbert, L. I. Insect Endocrinology. (Academic Press)). Without being bound by any one theory, phosphorylation of heterologously expressed FAR enzymes in yeast such as Y. lipolytica can lead to inactivation, and results in low fatty alcohol titers. In some embodiments, a bioinformatic approach can be used to predict phosphorylated residues within FAR. Alanine substitution of serine and threonine residues has been shown to abolish phosphorylation (Shi, S., Chen, Y., Siewers, V. & Nielsen, J. Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snf1-Dependent Regulation of Acc1. mBio 5 (2014)). Thus, the impact of alanine substitutions to prevent phosphorylation of serine residues and its impact on fatty alcohol titers can be tested. In addition to alanine substitution, improvement of FAR activity can also be achieved by other amino acid substitutions.

In some embodiments, methods are provided to identify beneficial mutations of FAR based on selection and alteration of phosphorylation-sensitive residues upon its expression in a host microorganism. In a preferred embodiment, the host microorganism is yeast selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, and Kluyveromyces.

Other references for protein phosphorylation sites include: Blom, N., Gammeltoft, S. & Brunak. S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites1. J. Mol. Biol. 294, 1351-1362 (1999); Ingrell, C. R., Miller, M. L., Jensen, O. N. & Blom, N. NetPhosYcast: prediction of protein phosphorylation sites in yeast. Bioinforma. 23: 895-897 (2007); Miller, W. T. Tyrosine kinase signaling and the emergence of multicellularity. Biochim. Biophys. Acta 1823, 1053-1057 (2012), each of which is herein incorporated in its entirety.

In some embodiments, a nucleic acid sequence encoding a fatty-acyl reductase from organisms of the species Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera is codon optimized. In some embodiments, the fatty acyl reductase comprises a nucleotide sequence set forth in SEQ ID NO: 1 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises a nucleotide sequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises a nucleotide sequence selected from SEQ ID NOs: 3, 32, 40, 72, 74, 76 and 81. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 77 from Yponomeuta evonymellus.

In some embodiments, the production of unsaturated fatty alcohols in a recombinant microorganism comprises the expression of one or more mutant FARs. In certain embodiments, Helicoverpa amigera fatty acyl-CoA reductase (HaFAR) variants are provided which have increased enzymatic activity relative to enzymatic activity of a wild type Helicoverpa amigera fatty acyl-CoA reductase encoded by an amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the increased enzymatic activity is a net activity increase in amount of fatty alcohol produced relative to the amount of fatty alcohol produced by a wild type enzymatic activity of HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, a wild type HaFAR comprises a nucleotide sequence set forth in SEQ ID NO: 90. In some embodiments, a variant of a wild type HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41 comprises point mutations at the following positions: S60X, S195X, S298X, S378X, S394X, S418X, and S453X, wherein X comprises an amino acid selected from F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W and R. In some embodiments, a variant of a wild type HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41 comprises a combination of point mutations selected from mutations at the following amino acid positions: S60X, S195X, S298X, S378X. S394X, S418X, and S453X, wherein X comprises an amino acid selected from F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W and R. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42-48. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises a nucleotide sequence selected from SEQ ID NOs: 83-89.

Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a fatty acyl reductase exhibiting at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58&, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of, 41, 42, 43, 44, 45, 46, 47, 48, 55, 56, 57, 73, 75, 77, and 82.

Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding for a fatty acyl reductase, wherein said nucleic acid molecule exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 1, 2, 3, 32, 37, 40, 72, 74, 76, 81, 83, 84, 85, 86, 87, 88, 89, and 90.

In some embodiments, the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding a fatty acyl reductase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to a fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.

Acyl-ACP Synthetase

The present disclosure describes enzymes that ligate a fatty acid to the corresponding fatty acyl-ACP.

In some embodiments, an acyl-ACP synthetase is used to catalyze the conversion of a fatty acid to a corresponding fatty acyl-ACP. An acyl-ACP synthetase is an enzyme capable of ligating a fatty acid to ACP to produce a fatty acid acyl-ACP. In some embodiments, an acyl-ACP synthetase can be used to catalyze the conversion of a fatty acid to a corresponding fatty acyl-ACP. In some embodiments, the acyl-ACP synthetase is a synthetase capable of utilizing a fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. In one such embodiment, a recombinant microorganism described herein can include a heterologous acyl-ACP synthetase, which catalyzes the conversion of a fatty acid to a corresponding fatty acyl-ACP. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule which encodes an acyl-ACP synthetase that catalyzes the conversion of a saturated C₆-C₂₄fatty acid to a corresponding saturated C₆-C₂₄fatty acyl-ACP.

Fatty Acid Synthase Complex

The present disclosure describes enzymes that catalyze the elongation of a carbon chain in fatty acid.

In some embodiments, a fatty acid synthase complex is used to catalyze initiation and elongation of a carbon chain in a fatty acid. A “fatty acid synthase complex” refers to a group of enzymes that catalyzes the initiation and elongation of a carbon chain on a fatty acid. The ACP along with the enzymes in the fatty acid synthase (FAS) pathway control the length, degree of saturation, and branching of the fatty acids produced. The steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (ace) gene families. Depending upon the desired product, one or more of these genes can be attenuated, expressed or over-expressed. In exemplary embodiments, one or more of these genes is over-expressed.

There are two principal classes of fatty acid synthases. Type I (FAS I) systems utilize a single large, multifunctional polypeptide and are common to both mammals and fungi (although the structural arrangement of fungal and mammalian synthases differ). The Type I FAS system is also found in the CMN group of bacteria (corynebacteria, mycobacteria, and nocardia). The Type 11 FAS (FAS II) is characterized by the use of discrete, monofunctional enzymes for fatty acid synthesis, and is found in archaca and bacteria.

The mechanism of FAS I and FAS II elongation and reduction is the substantially similar, as the domains of the FAS I multienzyme polypeptides and FAS II enzymes are largely conserved.

Fatty acids are synthesized by a series of decarboxylative Claisen condensation reactions from acetyl-CoA and malonyl-CoA. The steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (ace) gene families. For a description of this pathway, see, e.g., Heath et al. Prog. Lipid Res. 40:467, 2001, which is herein incorporated by reference in its entirety. Without being limited by theory, in bacteria, acetyl-CoA is carboxylated by acetyl-CoA carboxylase (Acc, a multi-subunit enzyme encoded by four separate genes, accABCD), to form malonyl-CoA. In yeast, acetyl-CoA is carboxylated by the yeast equivalents of the acetyl-CoA carboxylase, encoded by ACC1 and ACC2. In bacteria, the malonate group is transferred to ACP by malonyl-CoA:ACP transacylase (FabD) to form malonyl-ACP. In yeast, a malonyl-palmityl tranferase domain adds malonyl from malonyl-CoA to the ACP domain of the FAS complex. A condensation reaction then occurs, where malonyl-ACP merges with acyl-CoA, resulting in 1-ketoacyl-ACP. In this manner, the hydrocarbon substrate is elongated by 2 carbons.

Following elongation, the β-keto group is reduced to the fully saturated carbon chain by the sequential action of a keto-reductase (KR), dehydratase (DH), and enol reductase (ER). The elongated fatty acid chain is carried between these active sites while attached covalently to the phosphopantetheine prosthetic group of ACP. First, the β-ketoacyl-ACP is reduced by NADPH to form β-hydroxyacyl-ACP. In bacteria, this step is catalyzed by β-ketoacyl-ACP reductase (FabG). The equivalent yeast reaction is catalyzed by the ketoreductase (KR) domain of FAS. β-hydroxyacyl-ACP is then dehydrated to form trans-2-enoyl-ACP, which is catalyzed by either β-hydroxyacyl-ACP dehydratase/isomerase (FabA) or β-hydroxyacyl-ACP dehydratase (FabZ) in bacteria or the dehydratase (DH) domain of FAS in yeast. NADPH-dependent trans-2-enoyl-ACP reductase I, II, or III (Fabl, FabK, and FabL, respectively) in bacteria and the enol reductase (ER) domain of FAS in yeast reduces trans-2-enoyl-ACP to form acyl-ACP. Subsequent cycles are started by the condensation of malonyl-ACP with acyl-ACP by β-ketoacyl-ACP synthase I or β-ketoacyl-ACP synthase II (FabB and FabF, respectively, in bacteria or the beta-ketoacyl synthase (KS) domain in yeast).

In some embodiments, a fatty acid synthase complex can be used to catalyze elongation of a fatty acyl-ACP to a corresponding fatty acyl-ACP with a two carbon elongation relative to the substrate.

Dehydrogenase

The present disclosure describes enzymes that catalyze the conversion of a fatty aldehyde to a fatty alcohol. In some embodiments, an alcohol dehydrogenase (ADH, Table 3 and Table 3a) is used to catalyze the conversion of a fatty aldehyde to a fatty alcohol. A number of ADHs identified from alkanotrophic organisms. Pseudomonas fluorescens NRRL B-1244 (Hou et al. 1983), Pseudomonas butanovora ATCC43655 (Vangnai and Arp 2001), and Acinetobacter sp, strain M-1 (Tani et al. 2000), have shown to be active on short to medium-chain alkyl alcohols (C₂to C₁₄). Additionally, commercially available ADHs from Sigma, Horse liver ADH and Baker's yeast ADH have detectable activity for substrates with length C₁₀and greater. The reported activities for the longer fatty alcohols may be impacted by the difficulties in solubilizing the substrates. For the yeast ADH from Sigma, little to no activity is observed for C₁₂to C₁₄aldehydes by (Tani et al. 2000), however, activity for C₁₂and C₁₆hydroxy-ω-fatty acids has been observed (Lu et al. 2010). Recently, two ADHs were characterized from Geobacillus thermodenitrificans NG80-2, an organism that degrades C₁₅to C₃₆alkanes using the LadA hydroxylase. Activity was detected from methanol to 1-triacontanol (C₃₀) for both ADHs, with 1-octanol being the preferred substrate for ADH2 and ethanol for ADH1 (Liu et al. 2009).

The use of ADHs in whole-cell bioconversions has been mostly focused on the production of chiral alcohols from ketones (Ernst et al. 2005) (Schroer et al. 2007). Using the ADH from Lactobacillus brevis and coupled cofactor regeneration with isopropanol. Schroer et al. reported the production of 797 g of (R)-methyl-3 hydroxybutanoate from methyl acetoacetate, with a space time yield of 29 g/L/h (Schroer et al. 2007). Examples of aliphatic alcohol oxidation in whole-cell transformations have been reported with commercially obtained S. cerevisiae for the conversion of hexanol to hexanal (Presecki et al. 2012) and 2-heptanol to 2-heptanone (Cappaert and Larroche 2004).

TABLE 3

Exemplary alcohol dehydrogenase enzymes.

Organism
Gene Name
Accession No.

Bactrocera oleae (Olive fruit fly) (Dacus oleae)
ADH
Q9NAR7

Cupriavidus necator (Alcaligenes eutrophus)
adh
P14940

(Ralstonia eutropha)

Drosophila adiastola (Fruit fly) (Idiomyia
Adh
Q00669

adiastola)

Drosophila affinidisjuncta (Fruit fly) (Idiotnyia
Adh
P21518

affinidisjuncta)

Drosophila ambigua (Fruit fly)
Adh
P25139

Drosophila borealis (Fruit fly)
Adh
P48584

Drosophila differens (Fruit fly)
Adh
P22245

Drosophila equinoxialis (Fruit fly)
Adh
Q9NG42

Drosophila flavomontana (Fruit fly)
Adh
P48585

Drosophiia guanche (Fruit fly)
Adh
Q09009

Drsophila hawaiiensis (Fruit Fly)
Adh
P51549

Drosophila heteroneura (Fruit fly)
Adh
P21898

Drosophila immigrans (Fruit fly)
Adh
Q07588

Drosophila insulatis (Fruit fly)
Adh
Q9NG40

Drosophila lebanonensis (Fruit fly)
Adh
P10807

(Scaptodrosophila lebanonensis)

Drosophila mauritiana (Fruit fly)
Adh
P07162

Drosophila madeirensis (Fruit fly)
Adh
Q09010

Drosophila mimica (Fruit fly) (Idiomyia mimica)
Adh
Q00671

Drosophila nigra (Fruit fly) (Idiomyia nigra)
Adh
Q00672

Drosophila orena (Fruit fly)
Adh
P07159

Drosophila pseudoobscura bogotana (Fruit fly)
Adh
P84328

Drosophila picticornis (Fruit fly) (Idiomyia
Adh
P23361

picticornis)

Drosophila planitibia (Fruit fly)
Adh
P23277

Drosophila paulistorum (Fruit fly)
Adh
Q9U8S9

Drosophila silvestris (Fruit fly)
Adh
P23278

Drosophila subobscura (Fruit fly)
Adh
Q03384

Drosophila teissieri (Fruit fly)
Adh
P28484

Drosophila tsacasi (Fruit fly)
Adh
P51550

Fragaria ananassa (Strawberry)
ADH
P17648

Malus domestica (Apple) (Pyrus malus)
ADH
P48977

Scaptomyza albovittata (Fruit Fly)
Adh
P25988

Scaptomyza crassifemur (Fruit fly) (Drosophila
Adh
Q00670

crassifemur)

Sulfolobus sp. (strain RC3)
adh
P50381

Zaprionus tuberculatus (Vinegar fly)
Adh
P51552

Geobacilius stearothermophilus (Bacillus
adh
P42327

stearothermophilus)

Drosophila mayaguana (Fruit fly)
Adh, Adh2
P25721

Drosophila melanogaster (Fruit fly)
Adh, CG3481
P00334

Drosophila pseudoobscura (Fruit fly)
Adh, GA17214
Q6LCE4

Drosophila simulans (Fruit fly)
Adh, GD23968
Q24641

Drosophila yakuba (Fruit fly)
Adh, GE19037
P26719

Drophila ananassae (Fruit fly)
Adh, GF14888
Q50L96

Drosophila erecta (Fruit fly)
Adh, GG25120
P28483

Drosophila grimshawi (fruit fly) (Idiomyia
Adh, GH13025
P51551

grimshawi)

Drosophila willistoni (Fruit fly)
Adh, GK18290
Q05114

Drosophila persimilis Fruit fly)
Adh, GL25993
P37473

Drosophila sechellia (Fruit fly)
Adh, GM15656
Q9GN94

Cupriavidus necator (strain ATCC 17699/H16/
adh, H16_A0757
Q0KDL6

DSM 428/Stainer 337) (Ralstonia eutropha)

Mycobacterium tuberculosis (strain CDC 1551/
adh, MT1581
P9WQC2

Oshkosh)

Staphylococcus aureus (strain MW2)
adh, MT0568
Q8NXU1

Mycobacterium tuberculosis (strain ATCC 25618/
adh, Rv1530
P9WQC3

H37Rv)

Staphylococcus aureus (strain N315)
adh, SA0562
Q7A742

Staphylococcus aureus (strain bovine RF122/
adh, SAB0557
Q2YSX0

ET3-1)

Sulfolobus acidocaldarius (strain ATCC 33909/
adh, Saci_2057
Q4J781

DSM 639/JCM 8929/NBRC 15157/NCIMB

11770)

Staphylococcus aurcus (strain COL)
adh. SACOLO6
Q5H163

Staphylococcus aureus (strain NCTC 8325)
adh,
Q2G0G1

SAOUHSC_90608

Staphylococcus aureus (strain MRSA252)
adh, SAR0613
Q6GJ63

Staphylococcus aureus (strain MSSA476)
adh, SAS0573
Q6GBM4

Staphylococcus aureus (strain USA300)
adh,
Q2FJ31

SAUSA300_0594

Staphylococcus aureus (strain Mu50/ATCC
adh, SAV0605
Q99W07

700699)

Staphylococcus epidermidis (strain ATCC 12228)
adh. SE_0375
Q8CQ56

Staphylococcus epiderdis (strain ATCC 35984/
adh, SERP0257
Q5HRD6

RP62A)

Sulfolobus solfataricus (strain ATCC 35092/DSM
adh, SSO2536
P39462

1617/JCM 11322/P2)

Sulfolobus tokodaii (strain DSM 16993/JCM
adh, STK_25770
Q96XE0

10545 I NBRC 100140/7)

Anas platyrhynchos (Domestic duck) (Anas
ADH1
P30350

boschas)

Apteryx australis (Brown kiwi)
ADH1
P49645

Ceratitis capitata (Mediterranean fruitfly)
ADH1
P48814

(Tephritis capitata)

Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra)
ADH1
Q70UN9

Gallus (Chicken)
ADH1
P23991

Columba livia (Domestic pigeon)
ADH1
P86883

Coumix japonica (Japanese quail) (Coturnix
ADH1
P19631

japonica)

Drosophila hydei (Fruit fly)
Adh1
P23236

Drosophila montana (Fruit fly)
Adh1
P48586

Drosophila mettleri (Fruit fly)
Adh1
P22246

Drosophila mulleri (Fruit fly)
Adh1
P07161

Drosophila navojoa (Fruit fly)
Adh1
P12854

Geomys attwateri (Attwater's pocket gopher)
ADH1
Q9Z2M2

(Geomys bursarius attwateri)

Geomys bursarius (Plains pocket gopher)
ADH1
Q64413

Geomys knoxjonesi (Knox Jones's pocket gopher)
ADH1
Q64415

Hordeum vulgare (Barley)
ADH1
P05336

Kluyveromyces marxianus (Yeast) (candida kefyr)
ADH1
Q07288

Zea mays (Maize)
ADH1
P00333

Mesocricetus auratus (Golden hamster)
ADH1
P86885

Pennisetum americanum Pearl millet) (Pennisetum
ADH1
P14219

glaucum)

Petunia hybrida (Petunia)
ADH1
P25141

Oryctolagus cuniculus (Rabbit)
ADH1
Q03505

Solanum tuberosum (Potato)
ADH1
P14673

Struthio camelus (Ostrich)
ADH1
P80338

Trifolium repens (Creeping white clover)
ADH1
P13603

Zea luxurisns (Guaternalan teosinte) (Euchlaena
ADH1
Q07264

luxurians

Saccharomyces cerevisiae 'train ATCC204508/
ADH1, ADC1,
P00330

S288c) (Baker's yeast)
YOL:086C, O0947

Arabidopsis thaliana (Mouse-ear cress)
ADH1, ADH,
P06525

At1g77120,

F22K20.19

Schizosaccharomyces pombe (strain 972/ATCC
ADH1, adh,
P00332

24843) (Fission yeast)
SPCC13B11.01

Drosophiia lacicola (Fruit fly)
Adh1, Adh-1
027404

Mus musculus (Mouse)
Adh1, Adh-1
P00329

Peromyscus maniculatus (North American deer
ADH1, ADH-1
P41680

mouse)

Rattus norvegicus (Rat)
Adh1, Adh-1
P06757

Drosophila virilis (Fruit fly)
Adh1, Adh-1,
B4M8Y0

GJ18208

Scheffersomyces stipitis (strain ATCC 58785/
ADH1, ADH2,
O00097

CBS 6054/NBRC 10063/NRRL Y-11545)
PICST_68558

(Yeast) (Pichia stipitis)

Aspergillus flavus (strain ATCC 200026/FGSC
adh1,
P41747

A1120/NRRL 3357/JCM 12722/SRRC 167)
AFLA_048690

Neurospora crassa strain ATCC 24698/74-OR23-
adh-1,

1A/CBS 708.71/DSM 1257/FGSC 987)
B17C10.210,
Q9P6C8

NCU01754

Candida albicans (Yeast)
ADH1, CAD
P43067

Oryza sativa subsp. japonica (Rice)
ADH1, DUPR11.3,
Q2R8Z5

Os11g0210300,

LOC_Os11g10480,

OsJ_032001

Drosophila mojavensis (Fruit fly)
Adh1, GI17644
P09370

Kluyveromyces lactis strain ATCC 8585/CBS
ADH1,
P20369

2359/DSM 70799/NBRC 1267/NRRL Y-1140/
KLLA0F21010g

WM37) (Yeast) (Candida sphaerica)

Oryza sativa subsp. indica (Rice)
ADH1,
Q75ZX4

OsI_034290

Pongo abelii (Sumatran orangutan) (Pongo
ADH1A
Q5RBP7

pygmaeus abelii)

Homo sapiens (Human)
ADH1A, ADH1
P07327

Macaca mulatta (Rhesus macaque)
ADH1A, ADH1
P28469

Pan troglodytes (Chimpanzee)
ADH1B
Q5R1W2

Papio hamadryas (Hamadryas baboon)
ADH1B
P14139

Homo sapiens (Human)
ADH1B, ADH2
P00325

Homo sapiens (Human)
ADH1C, ADH3
P00326

Papio hamadiyas (Hamadryas baboon)
ADH1C, ADH3
O97959

Ceratitis capitata (Mediterranean fruit fly)
ADH2
P48815

(Tephritis capitata)

Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra)
ADH2
Q70UP5

Ceratitis rosa (Natal fruit fly) (Pterandrus rosa)
ADH2
Q70UP6

Drosophiia anizonae (Fruit fly)
Adh2
P27581

Drosophila buzzatii (Fruit fly)
Adh2
P25720

Drosophila hydei (Fruit fly)
Adh2
P23237

Drosophila montana (Fruit fly)
Adh2
P48587

Drosophila mulleri (Fruit fly)
Adh2
P07160

Drosophila wheeleri (Fruit fly)
Adh2
P24267

Entamoeba histolytica
ADH2
Q24803

Hordeum vulgare (Barley)
ADH2
P10847

Kluyveromyves marxianus (Yeast) (candida kefyr)
ADH2
Q9P4C2

Zea mays (Maize)
ADH2
P04707

Oryza sativa subsp. indica (Rice)
ADH2
Q4R1E8

Solarium lycopersicum (Tomato) (Lycopersicon
ADH2
P28032

esculentum)

Solanum iiiberosum (Potato)
ADH2
P14674

Scheffersomyces stipitis (strain ATCC 58785/
ADH2, ADH1,
O13309

CBS 6054/NBRC 10063/NRRL Y-11545)
PICST_27980

(Yeast) (Pichia stipitis)

Arabidopsis thaliana (Mouse-ear cress)
ADH2, ADHIII,
Q96533

FDH1, At5g43940,

MRH10.4

Saccharomyces cerevisiae (strain ATCC 204508/
ADH2, ADR2,

S288c) (Baker's yeast)
YMR303C,
P00331

YM9952.05C

Candida albicans (strain SC5314/ATCC MYA-
ADH2,
O94038

2876) (Yeast)
Ca41C10.04,

Ca019.12579,

Ca019.5113

Oryza sativa subsp. japonica (Rice)
ADH2, DUPR11.1
Q0ITW7

Os11g0210500,

LOC_Os11g10510

Drosophila mojavensis (Fruit fly)
Adh2, GI17643
P09369

Kluyveromyces lactis (strain ATCC 8585/CBS
ADH2,
P49383

2359/DSM 70799/NBRC 1267/NRRL Y-1140/
KLLA0F18260g

WM37) (Yeast) (Candida sphaerica)

Oryctolagus cuniculus (Rabbit)
ADH2-1
O46649

Oryctolagus cuniculus (Rabbit)
ADH2-2
O46650

Hordeum vulgare (Barley)
ADH3
P10848

Solanum tuberosum (Potato)
ADH3
P14675

Kluyveromyces lactis (strain ATCC 8585/CBS
ADH3,
P49384

2359/DSM 70799/NBRC 1267/NRRL Y-1140/
KLLA0B09064g

WM37) (Yeast) (Candida sphaerica)

Saccharomyces cerevisiae (strain ATCC 204508/
ADH3,
P07246

S288c) (Baker's yeast)
YMR083W,

YM9582.08

Homo sapiens (Human)
ADH4
P08319

Mus musculus (Mouse)
Adh4
Q9QYY9

Rattus norvegicus (Rat)
Adh4
Q64563

Struthio camelus (Ostrich)
ADH4
P80468

Kluyveromyces lactis (strain ATCC 8585/CBS
ADH4,
P49385

2359/DSM 70799/NBRC 1267/NRRL Y-1140/
KLLA0F13530g

WM37) (Yeast) (Candida sphaerica)

Schizosaccharomyces pombe (strain 972 ATCC
adh4,
Q09669

24843) (Fission yeast)
SPAC5H10.06c

Saccharomyces cerevisiae (strain YJM789)
ADH4, ZRG5,
A6ZTT5

(Baker's yeast)
SCY_1818

Saccharomyces cerevisiae (strain ATCC 204508/
ADH4, ZRG5,
P10127

S288c) (Baker's yeast)
YGL256W,

NRC465

Saccharomyces pastorianus (Lager yeast)
ADH5
Q6XQ67

(Saccharomyces cerevisiae x Saccharomyces

eubayanus)

Bos taurus (Bovine)
ADH5
Q3ZC42

Equus caballus (Horse)
ADH5
P19854

Mus musculus (Mouse)
Adh5, Adh-2,
P28474

Adh2

Rattus norvegicus (Rat)
Adh5, Adh-2,
P12711

Adh

Oryctolagus cuniculus (Rabbit)
ADH5, ADH3
O19053

Homo sapiens (Human)
ADH5, ADHX,
P11766

FDH

Dictyostelium discoideum (Slime mold)
adh5,
Q54TC2

DDB_G0281865

Saccharomyces cerevisiae (strain ATCC 204508/
ADH5,
P38113

S288c) (Baker's yeast)
YBR145W

YBR1122

Homo sapiens (Human)
ADH6
P28332

Peromyscus manic ulatus North American deer
ADH6
P41681

mouse)

Pongo abelii (Sumatran orangutan) (Pongo
ADH6
Q5R7Z8

pygmaeus abelii)

Rattus norvegicus (Rat)
Adh6
Q5X195

Homo sapiens (Human)
ADH7
P40394

Rattus norvegicus (Rat)
Adh7
P41682

Mus musculus (Mouse)
Adh7, Adh-3
Q64437

adh-3

Mycobacterium tuberculosis (strain CDC 1551/
adhA, MT1911
P9WQC0

Oshkosh)

Rkizohium meliloti (strain 1021) (Ensifer meliloti)
adhA, RA0704
O31186

(Sinorhizobium meliloti)
SMa1296

Mycobacterium tuberculosis (strain ATCC 25618/
adhA, Rv1862
P9WQC1

H37Rv)

Zymomonas mobilis subsp. mobilis (strain ATCC
adhA, ZMO1236
P20368

31821/ZM4/CP4)

Mycobacterium bovis (strain ATCC BAA-935/
adhB, Mb0784c
Q7U1B9

AF2122/97)

Mycobacterium tuberculosis (strain CDC 1551/
adhB, MT0786
P9WQC6

Oshkosh)

Mycobacterium tuberculosis (strain ATCC 25618/
adhB, Rv0761c,
P9WQC7

H37Rv)
MTCY369.06c

Zymomonas mobilis subsp. mobilis (strain ATCC
adhB, ZMO1596
P0DJA2

31821/ZM4/CP4)

Zymomonas mobilis subsp. mobilis (strain ATCC
adhB, Zmob_1541
F8DVL8

10988/DSM 424/LMG 404/NCIMB 8938/

NRRL B-806/ZM1)

Mycobacterium tuberculosis (strain CDC 1551/
adhD, MT3171
P9WQB8

Oshkosh)

Mycobacterium tuberculosis (strain ATCC 25618/
adhD, Rv3086
P9WQB9

H37Rv)

Clostridium acetobutylicum (strain ATCC 824/
adhE, aad,
P33744

DSM 792/JCM 1419/LMG 5710/VKM B-
CA_P0162

1787)

Escherichia coli (strain K12)
adhE, ana, b1241,
P0A9Q7

JW1228

Escherichia coli O157:H7
adhE, Z2016,
P0A9Q8

ECs1741

Rhodobacter sphaeroides (strain ATCC 17023/
adhI,
P72324

2.4.1/NCIB 8253/DSM 158)
RHOS4_11650,

RSP_2576

Oryza sativa subsp. indica (Rice)
ADI-IIII,
A2XAZ3

OsI_0009236

Escherichia coli (strain K12)
adhP, yddN,
P39451

b1478, JW1474

Geo-bacillus stearothermophilus (Bacillus
adhT
P12311

stearothermophilus)

Emericella nidulans (strain FGSC A4/ATCC
alcA, AN8979
P08843

38163/CBS 112.46/NRRL 194/M139)

(Aspergillus nidulans)

Emericella nidulans (strain FGSC A4/ATCC
alc, AN3741
P54202

38163/CBS 112.46/NRRL 194/M139)

(Aspergillus nidulans)

Emericella nidulans (strain FGSC A4/ATCC
alcC, adh3,
P07754

38163/CBS 112.46/NRRL 194/M139)
AN2286

(Aspergillus nidulans)

Arabidopsis thaliana (Mouse-ear cress)
At1g22430,
Q9SK86

F12K8.22

Arabidopsis thaliana (Mouse-ear cress)
At1g22440,
Q9SK87

F12K8.21

Arabidopsis thaliana (Mouse-ear cress)
At1g32780,
A1L4Y2

F6N18.16

Arabidopsis thaliana (Mouse-ear cress)
At1g64710,
Q8VZ49

F13011.3

Arabidopsis thaliana (Mouse-ear cress)
At4g22110
Q0V7W6

F1N20.210

Arabidopsis thaliana (Mouse-ear cress)
At5g24760,
Q8LEB2

T4C12_30

Arabidopsis thaliana (Mouse-ear cress)
At5g42250,
Q9FH04

K5.114.5

Zea mays (Maize)
FDH
P93629

Drosophila melanogaster (Fruit fly)
Fdh, gfd, ODH
P46415

CG6598

Bacillus subtilis (strain 168)
gbsB, BSU31050
P71017

Caenorhabditis elegans
H24K24.3
Q17335

Oryza sativa subsp. japonica (Rice)
Os02g0815500,
Q0DWH1

LOC_Os02g57040,

OsJ_008550,

P0643F09.4

Mycobacterium tuberculosis (strain ATCC 25618/
Rv1895
O07737

H37Rv)

Caenorhabditis elegans
sodh-1, K12G11.3
Q17334

Caenorhabditis elegans
sodh-2, K12G11.4
O45687

Pseudomonas sp.
terPD
P33010

Escherichia coli (strain K12)
yiaY, b3589,
P37686

JW5648

Moraxella sp. (strain TAE123)

P81786

Alligator mississippiensis (American alligator)

P80222

Catharanthus roseus (Madagascar periwinkle)

P85440

(Vinca rosea)

Gadus morhua subsp. callarias (Baltic cod) (Gadus

P26325

callarias)

Naja (Indian cobra)

P80512

Pisum sativum (Garden pea)

P12886

Pelophylax perez (Perez's frog) (Rana perezi)

P22797

Saara hardwickii (Indian spiny-tailedlizard)

P25405

(Uromastyx hardwickii)

Saara hardwickii (Indian spiny-tailed lizard)

P25406

(Uromastyx hardwickii)

Equus caballus (Horse)

P00327

Equus caballus (Horse)

P00328

Geobacillus stearothermophilus (Bacillus

P42328

stearothermophilus)

Gadus tnorhua (Atlantic cod)

P81600

Gad-us morhua (Atlantic cod)

P81601

Myxine glutinosa (Atlantic hagfish)

P80360

Octopus vulgaris (Common octopus)

P81431

Pisum sativum (Garden pea)

P80572

Saara hardwickii (Indian spiny-tailed lizard)

P80467

(Uromastryx hardwickii)

Scyliorhinus canicula (Small-spotted catshark)

P86884

(Squalus canicula)

Sparus aurata (Gilthead sea bream)

P79896

TABLE 3a

Additional Exemplary alcohol dehydrogenase enzymes

Organism
Accession No.

Helicoverpa armigera
A0A0176Q2K7

Helicoverpa armigera
A0A0F6Q2W6

Helicoverpa armigera
AKD01723.1

Helicoverpa amligera
A0A0F6Q4H2

Helicoverpa armigera
A0A0F6Q1E8

Helicoverpa armigera
A0A0F6Q2K3

Helicoverpa armigera
A0A0F6Q4H7

Helicoverpa armigera
A0A0F6Q2J9

Helicoverpa armigera
A0A0F6Q0W0

Helicoverpa armigera
A0A0F6Q0V0

Helicoverpa armigera
A0A0F6Q1F1

Helicoverpa armigera
A0A0F6Q2X2

Helicoverpa armigera
A0A0F6Q4I2

Helicoverpa armigera
A0A0F6Q2X0

Helicoverpa assulta
A0A0F6Q2L9

Helicoverpa assulta
A0A0F6Q4K1

Helicoverpa assulta
A0A0F6Q4J7

Helicoverpa assulta
A0A0F6Q2Y5

Helicoverpa assulta
A0A0F6Q2Y1

Helicoverpa assulta
A0A0F6Q1G6

Helicoverpa assulta
A0A0F6Q2Y9

Helicoverpa assulta
A0A0F6Q0X5

Helicoverpa assulta
A0A0F6Q2M3

Helicoverpa assulta
A0A0F6Q2L1

Helicoverpa assulta
A0A0F6Q1F9

Helicoverpa assulta
A0A0F6Q0W6

Helicoverpa assulta
A0A0F6Q1G9

Fielicoverpa assulta
A0A0F6Q2L4

Helicoverpa assulta
A0A0F6Q2X6

Helicoverpa assulta
A0A0F6Q1H3

Helicoverpa assulta
A0A0F6Q0X1

Bombyx mori
NP_001188510.1

Aedes aegypti
XP001655103 .1

Anopheles darlingi
ETN64198.1

Yarrowia lipolytica
YALI0F09603g (FADH),

YALI0D25630g (ADH1),

YALI0E17787g (ADH2),

YALI0A16379g (ADH3),

YALI0E15818g (ADH4),

YALI0D02167g (ADH5),

YALI0A15147g (ADH6),

YALI0E07766g (ADH7)

In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and or reduction in the activity of one or more endogenous (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7).

Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions or other disruptions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the proteins encoded by YALI0F09603g (FADH). YALI0D25630g (ADH1). YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), and YALI0E07766g (ADH7).

Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with uniprot database IDs Q6C297 (FADH). Q6C7T0 (ADH1), F2Z678 (ADH2), Q6CGT5 (ADH3), Q6C5R5 (ADH4), Q6CAT5 (ADH5), Q6CGX5 (ADH6), and Q6C7K3 (ADH7).

Alcohol Oxidase

The present disclosure describes enzymes that oxidize fatty alcohols to fatty aldehydes.

In some embodiments, an alcohol oxidase (AOX) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. Alcohol oxidases catalyze the conversion of alcohols into corresponding aldehydes (or ketones) with electron transfer via the use of molecular oxygen to form hydrogen peroxide as a by-product. AOX enzymes utilize flavin adenine dinucleotide (FAD) as an essential cofactor and regenerate with the help of oxygen in the reaction medium. Catalase enzymes may be coupled with the AOX to avoid accumulation of the hydrogen peroxide via catalytic conversion into water and oxygen.

Based on the substrate specificities, AOXs may be categorized into four groups: (a) short chain alcohol oxidase, (b) long chain alcohol oxidase, (c) aromatic alcohol oxidase, and (d) secondary alcohol oxidase (Goswami et al. 2013). Depending on the chain length of the desired substrate, some members of these four groups are better suited than others as candidates for evaluation.

Short chain alcohol oxidases (including but not limited to those currently classified as EC1.1.3.13, Table 4) catalyze the oxidation of lower chain length alcohol substrates in the range of C1-C8 carbons (van der Klei et al. 1991) (Ozimek et al. 2005). Aliphatic alcohol oxidases from methylotrophic yeasts such as Candida boidinii and Komagataella pastoris (formerly Pichia pastoris) catalyze the oxidation of primary alkanols to the corresponding aldehydes with a preference for unbranched short-chain aliphatic alcohols. The most broad substrate specificity is found for alcohol oxidase from the Pichia pastoris including propargyl alcohol, 2-chloroethanol, 2-cyanoethanol (Dienys et al. 2003). The major challenge encountered in alcohol oxidation is the high reactivity of the aldehyde product. Utilization of a two liquid phase system (water/solvent) can provide in-situ removal of the aldehyde product from the reaction phase before it is further converted to the acid. For example, hexanal production from hexanol using Pichia pastoris alcohol oxidase coupled with bovine liver catalase was achieved in a bi-phasic system by taking advantage of the presence of a stable alcohol oxidase in aqueous phase (Karra-Chaabouni et al. 2003). For example, alcohol oxidase from Pichia pastoris was able to oxidize aliphatic alcohols of C6 to C11 when used biphasic organic reaction system (Murray and Duff 1990). Methods for using alcohol oxidases in a biphasic system according to (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) are incorporated by reference in their entirety.

Long chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.20; Table 5) include fatty alcohol oxidases, long chain fatty acid oxidases, and long chain fatty alcohol oxidases that oxidize alcohol substrates with carbon chain length of greater than six (Goswami et al. 2013). Banthorpe et al. reported a long chain alcohol oxidase purified from the leaves of Tanacetum vulgare that was able to oxidize saturated and unsaturated long chain alcohol substrates including hex-trans-2-en-1-ol and octan-1-ol (Banthorpe 1976) (Cardemil 1978). Other plant species, including Simmondsia chinensis (Moreau, R. A., Huang 1979). Arabidopsis thaliana (Cheng et al. 2004), and Lotus japonicas (Zhao et al. 2008) have also been reported as sources of long chain alcohol oxidases. Fatty alcohol oxidases are mostly reported from yeast species (Hommel and Ratledge 1990) (Vanhanen et al. 2000) (Hommel et al. 1994) (Kemp et al. 1990) and these enzymes play an important role in long chain fatty acid metabolism (Cheng et al. 2005). Fatty alcohol oxidases from yeast species that degrade and grow on long chain alkanes and fatty acid catalyze the oxidation of fatty alcohols. Fatty alcohol oxidase from Candida tropicalis has been isolated as microsomal cell fractions and characterized for a range of substrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991) (Mauersberger et al. 1992). Significant activity is observed for primary alcohols of length C₈to C₁₆with reported K_Min the 10-50 μM range (Eirich et al. 2004). Alcohol oxidases described may be used for the conversion of medium chain aliphatic alcohols to aldehydes as described, for example, for whole-cells Candida boidinii (Gabelman and Luzio 1997), and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990). Long chain alcohol oxidases from filamentous fungi were produced during growth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha and Ratledge 1991). The long chain fatty alcohol oxidase (LjFAO1) from Lotus japonicas has been heterologously expressed in E. coli and exhibited broad substrate specificity for alcohol oxidation including 1-dodecanol and 1-hexadecanol (Zhao et al. 2008).

TABLE 4

Alcohol oxidase enzymes capable of oxidizing short chain alcohols (EC 1.1.3.13)

Organism
Gene names
Accession No.

Komagataella pastoris (strain ATCC 76273/
AOX1 PP7435_Chr4-
F2QY27

CBS 7435/CECT 11047/NRRL Y-11430/
0130

Wegner 21-1) (Yeast) (Pichia pastoris)

Komagataella pastoris (strain GS115/ATCC
AOX1
P04842

20864) (Yeast) (Pichia pastoris)
PAS_chr4_0821

Komagataella pastoris (strain ATCC 76273/
AOX2 PP7435_Chr4-
F2R038

CBS 7435/CECT 11047/NRRL Y-11430
0863

Wegner 21-1) (Yeast) (Pichia pastoris)

Komagataella pastoris (strain GS115/ATCC
AOX2
C4R702

20864) (Yeast) (Pichia pastoris)
PAS_chr4_0152

Candida boidinii (Yeast)
AOD1
Q00922

Pichia angusta (Yeast) (Hansenula polymorpha)
MOX
P04841

Thanatephorus cucumeris (strain AG1-IB/
AOD1 BN14_10802
M5CC52

isolate 7/3/14) (Lettuce bottom rot fungus)

(Rhizoctonia solani)

Thanatephoms cucumeris (strain AG1-IB/
MOX BN14_12214
M5CF32

isolate 7/3/14) (Lettuce bottom rot fungus)

(Rhizoctonia solani)

Thanatephonis cucumeris (strain AG1-IB/
AOD1 BN14_10691
M5CAV1

isolate 7/3/14) (Lettuce bottom rot fungus)

(Rhizoctonia solani)

Thanatephonis cucumeris (strain AG1-IB/
AOD1 BN14_09479
M5C7F4

isolate 7/3/14) (Lettuce bottom rot fungus)

(Rhizoctonia solani)

Thanatephoms cucumeris strain AG1-IB/
AOD1 BN14_10803
M5CB66

isolate 7/3/14) (Lettuce bottom rot fungus)

Rhizoctonia solani)

Thanatephorus cucutneris (strain AGI-IB/
AOD1 BN14_09900
M5C9N9

isolate 7/3/14) (Lettuce bottom rot fungus)

(Rhizoctonia. solani)

Thanatephorus cucumeris (strain AG1-IB/
AOD1 BN14_08302
M5C2L8

isolate 7/3/14) (Lettuce bottom rot fungus)

(Rhizoctonia solani)

Thanatephorus cucumeris (strain AG1-1B/
MOX BN14_09408
M5C784

isolate 7/3/14) (Lettuce bottom rot fungus)

Rhizoctonia solani)

Thanatephorus cucumeris (strain AG1-IB/
MOX BN14_09478
M5C8F8

isolate 7/3/14) (Lettuce bottom rot fungus)

(Rhizoctonia solani)

Thanatephorus cucumeris (strain AG1-IB/
AOD1 BN14_11356
M5CH40

isolate 7/3/14) (Lettuce bottom rot fungus)

(Rhizoctonia solani)

Ogataea henricii
AOD1
A5LGF0

Candida methanosorbosa
AOD1
A5LGE5

Candida methanolovescens
AOD1
A5LGE4

Candida succiphila
AOD1
A5LGE6

Aspergillus niger (strain CBS 513.88/FGSC
An18g05480
A2R501

A1513)

Aspergillus niger (strain CBS 513.88/FGSC
An18g05480
A2RB46

A1513)

Moniliophthora pemiciosa (Witches'-broom

17CMK2

disease fungus) (Marasmius pemiciosus)

Candida cariosilignicola
AOD1
A5LGE3

Candida pignaliae
AOD1
A5LGE1

Candida pignaliae
AOD2
A5LGE2

Candida sonorensis
AOD1
A5LGD9

Candida sonorensis
AOD2
A5LGE0

Pichia naganishii
AOD1
A5LGF2

Ogataea minuta
AOD1
A5LGF1

Ogataea philodendra
AOD1
A5LGF3

Ogataea wickerhamii
AOD1
A5LGE8

Kuraishia capsulate
AOD1
A5LGE8

Talaromyces stipitatus (strain ATCC 10500/
TSTA_021940
B8MHF8

CBS 375.48/QM 6759/NRRL 1006)

(Penicillium stipitatum)

Talaromyces stipitatus (strain ATCC 10500/
TSTA_065150
B8LTH7

CBS 375.48/QM 6759/NRRL 1006)

(Penicillium stipitatum)

Talaromyces stipitatus (strain ATCC 10500/
TSTA_065150
B8LTH8

CBS 375.48/QM 6759/NRRL 1006)

(Penicillium stipitatum)

Talaromyces stipitatus (strain ATCC 10500/
TSTA_000410
B8MSB1

CBS 375.48/QM 6759/NRRL 1006)

(Penicillium stipitatum)

Ogataea glucozyma
AOD1
A5LGE9

Ogataea parapolymorpha (strain DL-1/ATCC
HPODL_03886
W1QCJ3

26012/NRRL Y-7560) (Yeast) (Hansenula

polymorpha)

Gloeophyllum trabeum (Brown rot fungus)
AOX
A8DPS4

Pichia angusta (Yeast) (Hansenula polymorpha)
mox1
A6PZG8

Pichia trehalophila
AOD1
A5LGF4

Pichia angusta (Yeast) (Hansenula polymorpha)
mox1
A6PZG9

Pichia angusta (Yeast) (Hansenula polymorpha)
mox1
A6PZG7

Ixodes scapularis (Black-legged tick) (Deer
IscW_ISCW017898
B7PIZ7

tick)

TABLE 5

Alcohol oxidase enzymes capable of oxidizing long chain alcohols

including fatty alcohols (EC 1.1.3.20)

Accession

Organism
Gene names
No.

Lotus japonicus (Lotus
FAO1
B5WWZ8

corniculatus var.

japonicus)

Arabidopsis thaliana

FAO1 At1g03990
Q9ZWB9

(Mouse-ear cress)
F21M11.7

Lotus japonicus (Lotus
FAO2
B5WWZ9

corniculatus var.

japonicus)

Arabidopsis thaliana

FAO3 At3g23410
Q9LW56

(Mouse-ear cress)
MLM24.14

MLM24.23

Arabidopsis thaliana

FAO4A At4g19380
O65709

(Mouse-ear cress)
T5K18.160

Arabidopsis thaliana

FAO4B At4g28570
Q94BP3

(Mouse-ear cress)
T5F17.20

Microbotryum violaceum

MVLG_06864
U5HIL4

(strain p1A1

Lamole) (Anther smut

fungus) (Ustilago

violacea)

Ajellomyces dermatitidis

BIDFG_03507
T5BNQ0

ATCC 26199

Gibberella zeae (strain
FG06918.1
I1RS14

PH-1/ATCC
FGSG_06918

MYA-4620/FGSC

9075/NRRL

31084) (Wheat head

blight fungus)

(Fusarium graminearum)

Pichia sorbitophila

Piso0_004410
G8Y5E1

(strain ATCC MYA-
GNLVRS01_

4447/BCRC 22081/
PISO0K16268a

CBS 7064/

NBRC 10061/
GNLVRS01_

NRRLY-195)
PISO0L16269g

(Hybrid yeast)

Emericella nidulans

(strain FGSC A4/
AN0623.2
Q5BFQ7

ATCC 38163/CBS
AN1A_00623

112.46/NRRL 194/

M139) (Aspergillus

nidulans)

Pyrenophora tritici-

PTRG_10154
B2WJW5

repentis (strain Pt-

IC-BFP) (Wheat tan

spot fungus)

(Drechslera tritici-

repentis)

Paracoccidioides lutzii

PAAG_09117
C1HEC6

(strain ATCC

MYA-826/Pb01)

(Paracoccidioides

brasiliensis)

Candida parapsilosis

CPAR2_204420
G8BG15

(strain CDC 317/

ATCC MYA-4646)

(Yeast) (Monilia

parapsilosis)

Pseudozyma brasiliensis

PSEUBFA_
V5GPS6

(strain
SCAF2a03010

GHG001) (Yeast)

Candida parapsilosis

CPAR2_204430
G8BG16

(strain CDC 317/

ATCC MYA-4646)

(Yeast) (Monilia

parapsilosis)

Sclerotinia borealis

SBOR_5750
W9CDE2

F-4157

Sordaria macrospora

SMAC_06361
F7W6K4

(strain ATCC

MYA-333/DSM 997/

K(L3346)/K-

hell)

Sordaria macrospora

SMAC_01933
F7VSA1

(strain ATCC

MYA-333/DSM 997/

K(L3346)/K-

hell)

Meyerozyma

PGUG_03467
A5DJL6

guilliermondii

(strain ATCC 6260/CBS

566/DSM 6381/

JCM 1539/NBRC

10279/NRRL Y-

324) (Yeast) (Candida

guilliermiondii)

Trichophyton rubrum

H107_00669
A0A023ATC5

CBS 202.88

Arthrobotrys oligospora

AOL_00097g516
G1XJI9

(strain ATCC

24927/CBS 115.81/

DSM 1491)

(Nematode-trapping

fungus)

(Didymozoophaga

oligospora)

Scheffersomyces stipitis

FAO1 PICST_90828
A3LYX9

(strain ATCC

58785/CBS 6054/

NBRC 10063/

NRRL Y-11545) (Yeast)

(Pichia stipitis)

Scheffersomyces stipitis

FAO2 PICST_32359
A3LW61

(strain ATCC

58785/CBS 6054/

NBRC 10063/

NRRL Y-11545) (Yeast)

(Pichia stipitis)

Aspergillus oryzae

Ao304_09114
I8TL25

(strain 3.042)

(Yellow koji mold)

Fusarium oxysporum

FOXB_17532
F79GFU8

(strain Fo5176)

(Fusarium vascular wilt)

Rhizopus delemar

RO3G_08271
I1C536

(strain RA 99-880/

ATCC MYA-4621/

FGSC 9543/

NRRL 43880)

(Mucormycosis agent)

(Rhizopus arrhizus var.

delemar)

Rhizopus delemar

RO3G_00154
I1BGX0

(strain RA 99-880/

ATCC MYA-4621/

FGSC 9543/

NRRL 43880)

(Mucormycosis agent)

(Rhizopus arrhizus

var. delemar)

Fusarium oxysporum

FOXB_07532
F9FMA2

(strain Fo5176)

(Fusarium vascular wilt)

Penicillium roqueforti

PROQFM164_
W6QPY1

S02g001772

Aspergillus clavatus

ACLA_018400
A1CNB5

(strain ATCC 1007/

CBS 513.65/DSM 816/

NCTC 3887/

NRRL 1)

Arthrodemia otae (strain
MCYG_08732
C5G1B0

ATCC MYA-

4605/CBS 113480)

(Microsporum

canis)

Tiichophyton tonsurans

TESG_07214
F2S8I2

(strain CBS

112818) (Scalp ringworm

fungus)

Colletotrichum

CH063_13441
H1VUE7

higginsianum (strain IMI

349063) (Crucifer

anthracnose fungus)

Ajellomyces capsulatus

HCDG_07658
C6HN77

(strain H143)

(Darling's disease

fungus) (Histoplasma

capsulatum)

Trichophyton rubnim

TERG_08235
F2T096

(strain ATCC

MYA-4607/CBS

118892) (Athlete's

foot fungus)

Cochliobolus

COCHEDRAFT_
M2UMT9

heterostrophus (strain C5/
1201414

ATCC 48332/race O)

(Southern corn

leaf blight fungus)

(Bipolaris maydis)

Candida orthopsilosis

CORT_0D04510
H8X643

(strain 90-125)

(Yeast)

Candida orthopsilosis

CORT_0D04520
H8X644

(strain 90-125)

(Yeast)

Candida orthopsilosis

CORT_0D04530
H8X645

(strain 90-125)

(Yeast)

Pseudozyma aphidis

PaG_03027
W3VP49

DSM 70725

Coccidioides posadasii

CPC735_000380
C5P005

(strain C735)

(Valley fever fungus)

Magnaporthe oryzae

OOW_
L7IZ92

(strain P131) (Rice
P131scaffold01214g15

blast fungus) (Pyricularia

oryzae)

Neurospora tetrasperma

NEUTE1DRAFT_82541
F8MKD1

(strain FGSC

2508/ATCC MYA-

4615/P0657)

Hypocrea virens (strain
TRIVIDRAFT_54537
G9MMY7

Gv29-8/FGSC

10586) (Gliocladium

virens)

(Trichoderma virens)

Hypocrea virens (strain
TRIVIDRAFT_53801
G9MT89

Gv29-8/FGSC

10586) (Gliocladium

virens)

(Trichoderma virens)

Aspergillus niger (strain
An01g09620
A2Q9Z3

CBS 513.88/

FGSC A1513)

Verticillium dahliae

VDAG_05780
G2X6J8

(strain VdLs.17/

ATCC MYA-4575/

FGSC 10137)

(Verticillium wilt)

Ustilago maydis (strain
UM02023.1
Q4PCZ0

521/FGSC

9021) (Corn smut fungus)

Fusarium oxysporum f.

FOWG_13006
W9LNI9

sp. lycopersici

MN25

Fusarium oxysporum f.

FOWG_02542
W9N9Z1

sp. lycopersici

MN25

Candida tropicalis (Yeast)
FAO1
Q6QIR6

Magnaporthe oryzae

MGG_11317
G4MVK1

(strain 70-15/

ATCC MYA-4617/

FGSC 8958) (Rice

blast fungus) (Pyricularia

oryzae)

Candida tropicalis (Yeast)
faot
Q9P8D9

Candida tropicalis (Yeast)
FAO2a
Q6QIR5

Phaeosphaeria nodorum

SNOG_02371
Q0V0U3

(strain SN15/

ATCC MYA-4574/

FGSC 10173)

(Glume blotch fungus)

(Septoria

nodorum)

Candida tropicalis (Yeast)
FAO2b
Q6QIR4

Pestalotiopsis fici W106-1
PFICI_11209
W3WU04

Magnaporthe oryzae

OOU_
L7IFT5

(strain Y34) (Rice
Y34scaffold00240g57

blast fungus) (Pyricularia

oryzae)

Pseudogymnoascus

GMDG_01756
L8G0G6

destructans (strain

ATCC MYA-4855/

20631-21) (Bat

white-nose syndrome

fungus)

(Geomyces destructans)

Pseudogymnoascus

GMDG_04950
L8GCY2

destructans (strain

ATCC MYA-4855/

20631-21) (Bat

white-nose syndrome

fungus)

(Geomyces destructans)

Mycosphaerella fijiensis
MYCFIDRAFT_52380
M2Z831

(strain CIRAD86)

(Black leaf streak disease

fungus)

(Pseudocercospora

fijiensis)

Bipolaris oryzae

COCMIDRAFT_84580
W7A0I8

ATCC 44560

Cladophialophora

A1O5_08147
W9WTM9

psammophila CBS

110553

Fusarium oxysporum f.

FOMG_05173
X0AEE6

sp. melonis

26406

Fusarium oxysponim f.

FOMG_17829
W9ZBB7

sp. melonis

26406

Cyphellophora europaea

HMPREF1541_102174
W2S2S5

CBS 101466

Aspergillus kawachii

AKAW_00147
G7X626

(strain NBRC

4308) (White koji

mold) (Aspergillus

awamori var. kawachi)

Aspergillus terreus

ATEG_05086
Q0CMJ8

(strain NIH 2624/

FGSC A1156)

Coccidioides immitis

CIMG_02987
J3KAI8

(strain RS) (Valley

fever fungus)

Ajellomyces dermatitidis

BDCG_04701
C5GLS5

(strain ER-3/

ATCC MYA-2586)

(Blastomyces

dermatitidis)

Fusarium oxysporum f.

FOC1_g0013865
N4U732

sp. cubense

strain race 1) (Panama

disease fungus)

Rhodotorula glutinis

RTG_00643
G0SVU8

(strain ATCC

204091/IIP 30/MTCC

1151) (Yeast)

Aspergillus niger (strain
ASPNIDRAFT_35778
G3XTM6

ATCC 1015/

CBS 113.46/FGSC

A1144/LSHB Ac4/

NCTC 3858a/NRRL

328/USDA

3528.7)

Candida cloacae

fao1
Q9P8D8

Candida cloacae

fao2
Q9P8D7

Fusarium oxysporum f.

FOC1_g10006358
N4TUH3

sp. cubense

(strain race 1) (Panama

disease fungus)

Candida albicans

FAO1 CaO19.13562
Q59RS8

(strain SC5314/
orf19.13562

ATCC MYA-2876)

(Yeast)

Candida albicans

FAO1 CaO19.6143
Q59RP0

(strain SC5314/
orf19.6143

ATCC MYA-2876)

(Yeast)

Chaetomium

CTHT_0018560
G0S2U9

thermophilum

(strain DSM

1495/CBS 144.50/IMI

039719)

Mucor circinelloides f.

HMPREF1544_05296
S2JDN0

circinelloides

(strain 1006PhL)

(Mucormycosis

agent)

(Calyptromyces

circinelloides)

Mucor circinelloides

HMPREF1544_05295
S2JYP5

f. circinelloides

(strain 1006PhL)

(Mucormycosis agent)

(Calyptromyces

circinelloides)

Mucor circinelloides

HMPREF1544_06348
S2JVK9

f. circinelloides

(strain 1006PhL)

(Mucormycosis agent)

(Calyptromyces

circinelloides)

Botryotinia Fuckeliana

BCDW1_6807
M7UD26

(strain BcDW1)

(Noble rot fungus)

(Botrytis cinerea)

Podospora anserina

PODANS_5_13040
B2AFD8

(strain S/ATCC

MYA-4624/DSM 980/

FGSC 10383)

(Pleurage anserina)

Neosartorya fumigata

AFUA_1G17110
Q4WR91

(strain ATCC

MYA-4609/Af293/

CBS 101355/

FGSC A1100)

(Aspergillus

fumigatus)

Fusarium oxysporum f.

FOTG_00686
X0MEE6

sp. vasinfeetum

25433

Fusarium oxysporum f.

FOTG_12485
X0LE98

sp. vasinfectum

25433

Trichophyton interdigitale

H101_06625
A0A022U717

H6

Beauveria bassiana

BBA_04100
J4UNY3

(strain ARSEF 2860)

(White muscardine

disease fungus)

(Tritirachium shiotae)

Fusarium oxysporum f.

FOCG_00843
X0GQ62

sp. radicis-

lycopersici 26381

Fusarium oxysporum f.

FOCG_15170
X0F4T1

sp. radicis-

lycopersici 26381

Neurospora tetraspen

NEUTE2DRAFT_88670
G4UNN6

(strain FGSC

2509/P0656)

Pseudozyma hubeiensis

PHSY_000086
R9NVU1

(strain SY62)

(Yeast)

Lodderomyces

LELG_03289
A5E102

elongisporus (strain

ATCC 11503/CBS

2605/JCM 1781/

NBRC 1676/NRRL

YB-4239) (Yeast)

(Saccharomyces

elongisporus)

Malassezia globosa

MGL_3855
A8QAY8

(strain ATCC MYA-

4612/CBS 7966)

(Dandruff-associated

fungus)

Byssochlamys spectabilis

PVAR5_7014
V5GBL6

(strain No. 5/

NBRC 109023)

(Paecilomyces variotii)

Ajellomyces capsulatus

HCEG_03274
F0UF47

(strain H88)

(Darling's disease fungus)

(Histoplasma

capsulatum)

Trichosporon asahii

A1Q1_03669
J6FBP4

var. asahii (strain

ATCC 90039/CBS

2479/JCM 2466/

KCTC 7840/NCYC

2677/UAMH

7654) (Yeast)

Penicillium oxalicum

PDE_00027
S7Z8U8

(strain 114-2/

CGMCC 5302)

(Penicillium

decumbens)

Fusarium oxysporum f.

FOPG_02304
X0IBE3

sp. conglutinans

race 2 54008

Fusarium oxysporum f.

FOPG_13066
X0H540

sp. conglutinans

race 2 54008

Fusarium oxysporum f.

FOQG_00704
X0D1G8

sp. raphani

54005

Fusarium oxysporum f.

FOQG_10402
X0C482

sp. raphani

54005

Metarhizium acridum

MAC_03115
E9DZR7

(strain CQMa

102)

Arthroderma benhamiae

ARB_02250
D4B1C1

(strain ATCC

MYA-4681/CBS 112371)

(Trichophyton

mentagrophytes)

Fusarium oxysporum f.

FOIG_12161
X0JFI6

sp. cubense

tropical race 4 54006

Fusarium oxysporum f.

FOIG_12751
X0JDU5

sp. cubense

tropical race 4 54006

Cochliobolus

COCC4DRAFT_52836
N4WZZ0

heterostrophus

(strain C4/

ATCC 48331/race T)

(Southern corn

leaf blight fungus)

(Bipolaris maydis)

Trichosporon asahii

A1Q2_00631
K1VZW1

var. asahii (strain

CBS 8904) (Yeast)

Mycosphaerella

MYCGRDRAFT_37086
F9X375

graminicola (strain CBS

115943/IPO323)

(Speckled leaf blotch

fungus) (Septoria tritici)

Botryotinia fuckeliana

BofuT4_P072020.1
G2XQ18

(strain T4)

(Noble rot fungus)

(Botrytis cinerea)

Metarhizium anisopliae

MAA_05783
E9F0I4

(strain ARSEF

23/ATCC MYA-3075)

Cladophialophora

G647_J5801
V9DAR1

carrionii

CBS 160.54

Coccidioides posadasii

CPSG_09174
E9DH75

(strain RMSCC

757/Silveira) (Valley

fever fungus)

Rhodosporidium

RHTO_06879
M7X159

toruloides (strain

NP11) (Yeast)

(Rhodotorula gracilis)

Puccinia graminis f.

PGTG_10521
E3KIL8

sp. tritici (strain

CRL 75-36-700-3/race

SCCL) (Black

stem rust fungus)

Trichophyton rubrum

H103_00624
A0A022WG28

CBS 288.86

Colletotrichum

CFIO01_08202
A0A010RKZ4

fioriniae PJ7

Trichophyton rubrum

H104_00611
A0A022XB46

CBS 289.86

Cladophialophora

A1O7_02579
W9WC55

yegresii

CBS 114405

Colletotrichum orbiculare

Cob_10151
N4VFP3

(strain 104-T/

ATCC 96160/CBS

514.97/LARS 414/

MAFF 240422)

(Cucumber

anthracnose fungus)

(Colletotrichum

lagenarium)

Drechslerella

DRE_03459
W7IDL6

stenobrocha 248

Neosartorya fumigata

AFUB_016500
B0XP90

(strain CEA10/

CBS 144.89/

FGSC A1163)

(Aspergillus fumigatus)

Thielavia terrestris

THITE_2117674
G2R8H9

(strain ATCC 38088/

NRRL 8126)

(Acremonium

alabamense)

Gibberella fujikuroi

FFUJ_02948
S0D7P7

(strain CBS 19534/

IMI 58289/NRRL

A-6831) (Bakanae

and foot rot disease

fungus) (Fusarium

fujikuroi)

Gibberella fujikuroi

FFUJ_12030
S0EMC6

(strain CBS 195.34/

IMI 58289/NRRL

A-6831) (Bakanae

and foot rot disease

fungus) (Fusarium

fujikuroi)

Aspergillus flavus

AFLA_109870
B8N941

(strain ATCC 200026/

FGSC A1120/NRRL

3357/JCM

12722/SRRC 167)

Togninia minima

UCRPA7_1719
R8BTZ6

(strain UCR-PA7)

(Esca disease fungus)

(Phaeoacremonium

aleophilum)

Ajellomyces dermatitidis

BDDG_09783
F2TUC0

(strain ATCC

18188/CBS 674.68)

(Blastomyces

dermatitidis)

Macrophomina

MPH_10582
K2RHA5

phaseolina

(strain MS6)

(Charcoal rot fungus)

Neurospora crassa

NCU08977
Q7S2Z2

(strain ATCC 24698/

74-OR23-1A/CBS

708.71/DSM 1257/

FGSC 987)

Neosartorya fischeri

NFIA_008260
A1D156

(strain ATCC 1020/

DSM 3700/FGSC

A1164/NRRL

181) (Aspergillus

fischerianus)

Fusarium

FPSE_11742
K3U9J5

pseudograminearum

(strain

CS3096) (Wheat and

barley crown-rot

fungus)

Spathaspora

SPAPADRAFT_54193
G3AJP0

passalidarum

(strain NRRL

Y-27907/11-Y1)

Spathaspora

SPAPADRAFT_67198
G3ANX7

passalidarum

(strain NRRL

Y-27907/11-Y1)

Trichophyton verrucosum

TRV_07960
D4DL86

(strain HKI

0517)

Arthroderma gypseum

MGYG_07264
E4V2J0

(strain ATCC

MYA-4604/CBS 118893)

(Microsporum gypseum)

Hypocrea jecorina

TRIREDRAFT_43893
G0R7P8

(strain QM6a)

(Trichoderma reesei)

Trichophyton rubrum

H110_00629
A0A022Z1G4

MR1448

Aspergillus ruber CBS
EURHEDRAFT_512125
A0A017SPR0

135680

Glarea lozoyensis (strain
GLAREA_04397
S3D6C1

ATCC 20868/

MF5171)

Setosphaeria turcica

SETTUDRAFT_20639
R0K6H8

(strain 28A)

(Northern leaf

blight fungus)

(Exserohilum turcicum)

Paracoccidioides

PADG_0655
C1GH16

brasiliensis (strain

Pb18)

Fusarium oxysporum

FOZG_13577
W9JPG9

Fo47

Fusarium oxysporum

FOZG_05344
W9KPH3

Fo47

Trichophyton rubrum

H113_00628
A0A022ZY09

MR1459

Penicillium mameffei

PMAA_075740
B6QRY3

(strain ATCC

18224/CBS 334.59/

QM 7333)

Sphaerulina musiva

SEPMUDRAFT_154026
M3DAK6

(strain SO2202)

(Poplar stem canker

fungus) (Septoria

musiva)

Gibberella moniliformis

FVEG_10526
W7N4P8

(strain M3125/

FGSC 7600) (Maize

ear and stalk rot

fungus) (Fusarium

verticillioides)

Gibberella moniliformis

FVEG_08281
W7MVR9

(strain M3125/

FGSC 7600) (Maize

ear and stalk rot

fungus) (Fusarium

verticillioides)

Pseudozyma antarctica

PANT_22d00298
M9MGF2

(strain T-34)

Yeast) (Candida

amarctica)

Paracoccidioides

PBG_07795
C0SJD4

brasiliensis (strain

Pb03)

Rhizophagus irregularis

GLOINDRAFT_82554
U9TF61

(strain DAOM

181602/DAOM

197198/MUCL

43194) (Arbuscular

mycorrhizal fungus)

(Glomus intraradices)

Penicillium chrysogenum

Pc21g23700
B6HJ58

(strain ATCC
PCH_Pc21g23700

28089/DSM 1075/

Wisconsin 54-

1255) (Penicillium

notatum)

Baudoinia compniacensis

BAUCODRAFT_274597
M2M6Z5

(strain UAMH

10762) (Angels'

share fungus)

Hypocrea atroviridis

TRIATDRAFT_280929
G9NJ32

(strain ATCC

20476/IMI 206040)

(Trichoderma

atroviride)

Colletotrichum

CGLO_06642
T0LPH0

gloeosporioides (strain

Cg-14) (Anthracnose

fungus)

(Glomerella cingulata)

Cordyceps militaris

CCM_02665
G3JB34

(strain CM01)

(Caterpillar fungus)

Pyronema omphalodes

PCON_13062
U4LKE9

(strain CBS

100304) (Pyronema

confluens)

Colletotrichum

GLRG_08499
E3QR67

graminicola (strain

M1.001/M2/FGSC

10212) (Maize

anthracnose fungus)

(Glomerella

graminicola)

Glarea lozoyensis

M7I_2117
H0EHX4

(strain ATCC 74030/

MF5533)

Fusarium oxysporum f.

FOC4_g10002493
N1S969

sp. cubense

(strain race 4) (Panama

disease fungus)

Fusarium oxysporum f.

FOC4_g10011461
N1RT80

sp. cubense

(strain race 4) (Panama

disease fungus)

Cochliobolus sativus

COCSADRAFT_295770
M2TBE4

(strain ND90Pr/

ATCC 201652)

(Common root rot and

spot blotch fungus)

(Bipolaris

sorokiniana)

Mixia osmundae

Mo05571 E5Q_05571
G7E7S3

(strain CBS 9802/

IAM 14324/JCM

22182/KY 12970)

Mycosphaerella pini

DOTSEDRAFT_69651
N1PXR0

(strain NZE10/

CBS 128990) (Red

band needle blight

fungus) (Dothistroma

septosporum)

Grosmannia clavigera

CMQ_1113
F0XC64

(strain kw1407/

UAMH 11150) (Blue

stain fungus)

(Graphiocladiella

clavigera)

Fusarium oxysporum

FOYG_03004
W9IUE5

FOSC 3-a

Fusarium oxysporum

FOYG_16040
W9HNP0

FOSC 3-a

Fusarium oxysporum

FOYG_17058
W9HB31

FOSC 3-a

Nectria haematococca

NECHADRAFT_37686
C7YQL1

(strain 77-13-4/

ATCC MYA-4622/

FGSC 9596/

MPVI) (Fusarium solani

subsp. pisi)

Nectria haematococca

NECHADRAFT_77262
C7ZJI0

(strain 77-13-4/

ATCC MYA-4622/

FGSC 9596/

MPVI) (Fusarium solani

subsp. pisi)

Tuber melanosporum

GSTUM_00010376001
D5GLS0

(strain Me128)

(Perigord black truffle)

Ajellomyces dermatitidis

BDBG_07633
C5JYI9

(strain SLH14081)

(Blastomyces dermatitidis)

Chaetomium cdobosum

CHGG_09885
Q2GQ69

(strain ATCC

6205/CBS 148.51/

DSM 1962/NBRC

6347/NRRL 1970)

(Soil fungus)

Candida tenuis

CANTEDRAF_1108652
G3B9Z1

(strain ATCC 10573/

BCRC 21748/CBS

615/JCM 9827/

NBRC 10315/NRRL

Y-1498/VKM

Y-70) (Yeast)

Trichophyton rubrum

H102_00622
A0A022VKY4

CBS 100081

Pyrenophora teres f.

PTT_09421
E3RLZ3

teres (strain 0-1)

(Barley net blotch

fungus) (Drechslera

teres f. teres)

Colletotrichum

CGGC5_4608
L2GB29

gloeosporioides (strain

Nara gc5)

(Anthracnose fungus)

(Glomerella cingulata)

Gibberella zeae

FG05_06918
A0A016PCS4

(Wheat head blight

fungus) (Fusarium

graminearum)

Trichophyton soudanense

H105_00612
A0A022Y6A6

CBS 452.61

Sclerotinia sclerotiorum

SS1G_07437
A7EQ37

(strain ATCC

18683/1980/Ss-1)

(White mold)

(Whetzelinia

sclerotiorum)

Fusarium oxysporum f.

FOVG_14401
W9NWU8

sp. pisi HDV247

Fusarium oxysporum f.

FOVQ_02874
W9Q5V3

sp. pisi HDV247

Ustilago hordei

UHOR_03009
12G1Z4

(strain Uh4875-4)

(Barley covered smut

fungus)

Sporisorium reilianum

sr12985
E6ZYF7

(strain SRZ2)

(Maize head smut fungus)

Bipolaris zeicola 26-R-13
COCCADRAFT_81154
W6YIP8

Melampsora larici-

MELLADRAFT_78490
F4RUZ8

populina (strain

98AG31/pathotype 3-4-7)

(Poplar leaf

rust fungus)

Fusarium oxysporum f.

FOXG_01901
J9MG95

sp. lycopersici

(strain 4287/CBS

123668/FGSC 9935/

NRRL 34936) (Fusarium

vascular wilt

of tomato)

Fusarium oxysporum f.

FOXG_11941
J9N9S4

sp. lycopersici

(strain 4287/CBS

123668/FGSC 9935/

NRRL 34936) (Fusarium

vascular wilt

of tomato)

Bipolaris victoriae FI3
COCVIDRAFT_39053
W7EMJ8

Debaryomyces hansenii

DEHA2E04268g
Q6BQL4

(strain ATCC

36239/CBS 767/JCM

1990/NBRC

0083/IGC 2968)

(Yeast) (Torulaspora

hansenii)

Clavispora lusitaniae

CLUG_01505
C4XZX3

(strain ATCC

42720) (Yeast) (Candida

lusitaniae)

Candida albicans (strain
CAWG_02023
C4YME4

WO-1) (Yeast)

Trichophyton rubrum

H100_00625
A0A022U0Q2

MR850

Candida dubliniensis

CD36_32890
B9WMC7

(strain CD36/

ATCC MYA-646/CBS

7987/NCPF

3949/NRRL Y-17841)

(Yeast)

Starmerella bombicola

AOX1
A0A024FB95

Thielavia heterothallica

MYCTH_103590
G2QJL7

(strain ATCC

42464/BCRC 31852/

DSM 1799)

(Myceliophthora

thermophila)

Claviceps purpurea

CPUR_07614
M1WFI4

(strain 20.1) (Ergot

fungus) (Sphacelia

segetum)

Aspergillus oryzae

AO090023000571
Q2UH61

(strain ATCC 42149/

RIB 40) (Yellow

koji mold)

Dictyostelium discoideum

DDB_0184181
Q54DT6

(Slime mold)
DDB_G0292042

Triticum urartu

TRIUR3_22733
M7YME5

(Red wild einkorn)

(Crithodium urartu)

Solanum tuberosum

PGSC0003DMG400017211
M1BG07

(Potato)

Oryza saliva subsp.
OSJNBb0044B19.5
Q8W5P8

japonica (Rice)
LOC_Os10g33540

Oryza sativa subsp.
OJ1234_B11.20
Q6K9N5

japonica (Rice)
Os02g0621800

Oryza sativa subsp.
OSJNBa0001K12.5
Q8W5P3

japonica (Rice)
LOC_Os10g33520

Zea mays (Maize)
ZEAMMTB73_809149
C0P3J6

Citrus clementina

CICLE_v10011111mg
V4S9P4

Citrus clementina

CICLE_v10018992mg
V4U4C9

Citrus clementina

CICLE_v10004405mg
V4S9D3

Citrus clementina

CICLE_v10004403mg
V4R2Z6

Morus notabilis

L484_011703
W9RIK0

Morus notabilis

L484_005930
W9RET7

Medicago truncatula

MTR_1g075650
G7I4U3

(Barrel medic)

(Medicago tribuloides)

Arabidopsis thaliana

Q8LDP0

(Mouse-ear cress)

Medicago truncatula

MTR_4g081080
G7JF07

(Barrel medic)

(Medicago tribuloides)

Simmondsia chinensis

L7VFV2

(Jojoba) (Buxus

chinensis)

Prunus persica (Peach)
PRUPE_ppa018458mg
M5VXL1

(Amygdalus

persica)

Aphanomyces astaci

H257_07411
W4GI89

Aphanomyces astaci

H257_07412
W4GI44

Aphanomyces astaci

H257_07411
W4GKE3

Aphanomyces astaci

H257_07411
W4GK29

Aphanomyces astaci

H257_07411
W4GJ79

Aphanomyces astaci

H257_07411
W4GI38

Phaeodactylum

PHATRDRAFT_48204
B7G6C1

tricornutum (strain

CCAP 1055/1)

Hordeum vulgare

F2E4R4

var. distichum (Two-

rowed barley)

Hordeum vulgare

F2DZG1

var. distichum (Two-

rowed barley)

Hordeum vulgare

M0YPG7

var. distichum (Two-

rowed barley)

Hordeum vulgare

M0YPG6

var. distichum (Two-

rowed barley)

Hordeum vulgare

F2CUY4

var. distichum (Two-

rowed barley)

Ricinus communis

RCOM_0867830
B9S1S3

(Castor bean)

Brassica rapa subsp.
BRA014947
M4DEM5

pekinensis (Chinese

cabbage) (Brassica

pekinensis)

Ricinus communis

RCOM_0258730
B9SV13

(Castor bean)

Brassica rapa subsp.
BRA001912
M4CCI2

pekinensis (Chinese

cabbage) (Brassica

pekinensis)

Brassica rapa subsp.
BRA012548
M4D7T8

pekinensis (Chinese

cabbage) (Brassica

pekinensis)

Brassica rapa subsp.
BRA024190
M4E5Y6

pekinensis (Chinese

cabbage) (Brassica

pekinensis)

Brassica rapa subsp.
BRA015283
M4DFL0

pekinensis (Chinese

cabbage) (Brassica

pekinensis)

Ricinus communis

RCOM_1168730
B9SS54

(Castor bean)

Zea mays (Maize)

C4J691

Oryza glaberrima

I1P2B7

(African rice)

Zea mays (Maize)

B6SXM3

Zea mays (Maize)

C0HFU4

Aegilops tauschii

F775_19577
R7W4J3

(Tausch's goatgrass)

(Aegilops squarrosa)

Solanum habrochaites

R9R6T0

(Wild tomato)

(Lycopersicon hirsutum)

Physcomitrella patens

PHYPADRAFT_124285
A9S535

subsp. patens

(Moss)

Physcomitrella patens

PHYPADRAFT_113581
A9RG13

subsp. patens

(Moss)

Physcomitrella patens

PHYPADRAFT_182504
A9S9A5

subsp. patens

(Moss)

Solarium pennellii

R9R6Q1

(Tomato)

(Lycopersicon pennellii)

Vitis vinifera (Grape)
VIT_02s0087g00630
F6HJ27

Vitis vinifera (Grape)
VIT_07s0005g03780
F6HZM3

Vitis vinifera (Grape)
VIT_05s0049g01400
F6H8T4

Vitis vinifera (Grape)
VITISV_019349
A5AH38

Capsella rubella

CARUB_v10013046mg
R0HIT3

Capsella rubella

CARUB_v10004212mg
R0GUX4

Capsella rubella

CARUB_v10004208mg
R0F3X6

Capsella rubella

CARUB_v10012453mg
R0ILD0

Capsella rubella

CARUB_v10004208mg
R0GUX1

Eutrema salsugineum

EUTSA_v10024496mg
V4MD54

(Saltwater cress)

(Sisymbrium salsugineum)

Eutrema salsugineum

EUTSA_v10020141mg
V4NM59

(Saltwater cress

(Sisymbrium salsugineum)

Eutrema salsugineum

EUTSA_v10024496mg
V4LUR9

(Saltwater cress

(Sisymbrium salsugineum)

Eutrema salsugineum

EUTSA_v10024528mg
V4P767

(Saltwater cress)

(Sisymbrium salsugineum)

Eutrema salsugineum

EUTSA_v10006882mg
V4L2P6

(Saltwater cress

(Sisymbrium salsugineum)

Selaginella moellendorffii

SELMODRAFT_87684
D8R6Z6

(Spikemoss)

Selaginella moellendorffii

SELMODRAFT_87621
D8R6Z5

(Spikemoss)

Selaginella moellendorffii

SELMODRAFT_74601
D8QN81

(Spikemoss)

Selaginella moellendorffii

SELMODRAFT_73531
D8QN82

(Spikemoss)

Sorghum bicolor
Sb04g026390
C5XXS4

(Sorghum)

(Sorghum
SORBIDRAFT_04g026390

vulgare)

Sorghum bicolor
Sb04g026370
C5XXS1

(Sorghum)

(Sorghum
SORBIDRAFT_04g026370

vulgare)

Sorghum bicolor
Sb01g019470
C5WYH6

(Sorghum)

(Sorghum
SORBIDRAFT_01g019470

vulgare)

Sorghum bicolor
Sb0lg019480
C5WYH7

(Sorghum)

(Sorghum
SORBIDRAFT_01g019480

vulgare)

Sorghum bicolor
Sb01g019460
C5WYH5

(Sorghum)

(Sorghum
SORBIDRAFT_01g019460

vulgare)

Solanum pimpinellifolium

R9R6J2

(Currant

tomato) (Lycopersicon

pimpinellifolium)

Phaseolus vulgaris

PHAVU_007G124200g
V7BGM7

(Kidney bean)

(French bean)

Phaseolus vulgaris

PHAVU_011G136600g
V7AI35

(Kidney bean)

(French bean)

Phaseolus vulgaris

PHAVU_001G162800g
V7D063

(Kidney bean)

(French bean)

Solanum tuberosum

PGSC0003DMG400024294
M1C923

(Potato)

Solanum tuberosum

PGSC0003DMG400018458
M1BKV4

(Potato)

Solanum tuberosum

PGSC0003DMG400018458
M1BKV3

(Potato)

Glycine max (Soybean)

K7LK61

(Glycine hispida)

Glycine max (Soybean)

K7KXQ9

(Glycine hispida)

Populus trichocarpa

POPTR_0008s16920g
B9HKS3

(Western balsam

poplar) (Populus

balsamifera subsp.

trichocarpa)

Picea sitchensis

B8LQ84

(Sitka spruce) (Pinus

sitchensis)

Populus trichocarpa

POPTR_0004s24310g
U5GKQ5

(Western balsam

poplar) (Populus

balsamifera subsp.

trichocarpa)

Populus trichocarpa

POPTR_0010s07980g
B9HSG9

(Western balsam

poplar) (Populus

balsamifera subsp.

trichocarpa)

Glycine max (Soybean)

I1N9S7

(Glycine hispida)

Glycine max (Soybean)

I1LSK5

(Glycine hispida)

Setaria italica (Foxtail
Si034362m.g
K4A658

millet) (Panicum

italicum)

Solanum lycopersicum

Solyc09g072610.2
K4CUT7

(Tomato)

(Lycopersicon esculentum)

Setaria italica (Foxtail
Si016380m.g
K3YQ38

millet) (Panicum

italicum)

Solanum lycopersicum

R9R6I9

(Tomato)

(Lycopersicon esculentum)

Solanum lycopersicum

Solyc09g090350.2
K4CW61

(Tomato)

(Lycopersicon esculentum)

Solarium lycopersicum

Solyc08g005630.2
K4CI54

(Tomato)

(Lycopersicon esculentum)

Solanum lycopersicum

Solyc08g075240.2
K4CMP1

(Tomato)

(Lycopersicon esculentum)

Setaria italica (Foxtail
Si034359m.g
K4A655

millet) (Panicum

italicum)

Setaria italica (Foxtail
Si034354m.g
K4A650

millet) (Panicum

italicum)

Mimulus guttatus (Spotted
MIMGU_mgv1a001896mg
A0A022PU07

monkey flower)

(Yellow monkey flower)

Mimulus guttatus

MIMGU_mgv1a022390mg
A0A022RAV4

(Spotted monkey

flower) (Yellow

monkey flower)

Mimulus guttatus

MIMGU_mgv1a001868mg
A0A022S2E6

(Spotted monkey

flower) (Yellow

monkey flower)

Mimulus guttatus

MIMGU_mgv1a001883mg
A0A022S275

(Spotted monkey

flower) (Yellow

monkey flower)

Mimulus guttatus

MIMGU_mgv1a001761mg
A0A022QNF0

(Spotted monkey

flower) (Yellow

monkey flower)

Musa acuminata

M0SNA8

subsp. malaccensis

(Wild banana)

(Musa malaccensis)

Musa acuminata

M0RUT7

subsp. malaccensis

(Wild banana)

(Musa malaccensis

Musa acuminata

M0RUK3

subsp. malaccensis

(Wild banana)

(Musa malaccensis)

Saprolegnia diclina VS20
SDRG_10901
T0RG89

Brachypodium distachyon

BRADI3G49085
I1IBP7

(Purple false

brome) (Trachynia

distachya)

Brachypodium distachyon

BRADI3G28677
I1I4N2

(Purple false

brome) (Trachynia

distachya)

Brachypodium distachyon

BRADI3G28657
I1I4N0

(Purple false

brome) (Trachynia

distachya)

Oryza saliva subsp.
OsI_34012
B8BHG0

indica (Rice)

Oryza sativa subsp.
OsI_08118
B8AFT8

indica (Rice)

Orvza saliva subsp.
OsI_34008
A2Z8H1

indica (Rice)

Oryza sativa subsp.
OsI_34014
B8BHG1

indica (Rice)

Oryza sativa subsp.
LOC_Os10g33460
Q7XDG3

japonica (Rice)

Oryza sativa subsp.
Os10g0474800
Q0IX12

japonica (Rice)

Oryza saliva subsp.
0s10g0474966
C7J7R1

japonica (Rice)

Oryza saliva subsp.
OSJNBa0001K12.13
Q8W5N7

japonica (Rice)

Oryza sativa subsp.
OsJ_31873
B9G683

japonica (Rice)

Oryza sativa subsp.
OsJ_31875
B9G684

japonica (Rice)

Oryza sativa subsp.
OSJNBa0001K12.3
Q8W5P5

japonica (Rice)

Arabidopsis lyrata subsp.
ARALYDRAFT_470376
D7KDA3

lyrata (Lyre-

leaved rock-cress)

Arabidopsis lyrata subsp.
ARALYDRAFT_479855
D7L3B6

lyrata (Lyre-

leaved rock-cress)

Arabidopsis lyrata subsp.
ARALYDRAFT_491906
D7MDA9

lyrata (Lyre-

leaved rock-cress)

Arabidopsis lyrata subsp.
ARALYDRAFT_914728
D7MGS9

lyrata (Lyre-

leaved rock-cress)

Acetyl Transferase

The present disclosure describes enzymes that convert alcohols to fatty acetates.

In some embodiments, an acetyl transferase is used to catalyze the conversion of a fatty alcohol to a fatty acetate. An acetyl transferase is an enzyme that has the ability to produce an acetate ester by transferring the acetyl group from acetyl-CoA to an alcohol. In some embodiments, the acetyl transferase may have an EC number of 2.3.1.84.

The acetyl transferase, or the nucleic acid sequence that encodes it, can be isolated from various organisms, including but not limited to, organisms of the species Candida glabrata, Saccharomyces cerevisiae, Danaus plexippus, Heliotis virescens, Bombyx mori, Agrotis ipsilon, Agrotis segetum, Euonymus alatus, Homo sapiens, Lachancea thermotolerans and Yarrowia lipolytica. In exemplary embodiments, the acetyl transferase comprises a sequence selected from GenBank Accession Nos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061, KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, and XP_505595.1, and XP_505513.1. Exemplary acetyl transferase enzymes are listed in Table 5d. Additional exemplary acetyl transferase peptides may be found in US2010/0199548, which is herein incorporated by reference.

TABLE 5d

Exemplary acetyl transferase enzymes

Organism
Enzyme ID

Candida glabarata

KTA99184.1

Agrotis segetum

AIN34693.1

Homo sapiens

AY605053

Lachancea thermotolerans

XP_002552712.1

Yarrowia lipolytica

XP_503024.1

Yarrowia lipolytica

XP_505595.1

Yarrowia lipolytica

XP_505513.1

Fatty Acyl-ACP Thioesterase

Acyl-ACP thioesterase releases free fatty acids from Acyl-ACPs, synthesized from de novo fatty acid biosynthesis. The reaction terminates fatty acid biosynthesis. In plants, fatty acid biosynthesis occurs in the plastid and thus requires plastid-localized acyl-ACP thioesterases. The main products of acyl-ACP thioesterase are oleate (C18:0) and to a lesser extent palmitate (C16:0) in the vegetative tissues of all plants. The released free fatty acids are re-esterified to coenzyme A in the plastid envelope and exported out of plastid.

There are two isoforms of acyl-ACP thioesterase, FatA and FatB. Substrate specificity of these isoforms determines the chain length and level of saturated fatty acids in plants. The highest activity of FatA is with C18:1-ACP. FatA has very low activities towards other acyl-ACPs when compared with C18:1-ACP. FatB has highest activity with C16:0-ACP. It also has significant high activity with C18:1-ACP, followed by C18:0-ACP and C16:1-ACP. Kinetics studies of FatA and FatB indicate that their substrate specificities with different acyl-ACPs came from the Kcat values, rather than from Km. Km values of the two isoforms with different substrates are similar, in the micromolar order. Domain swapping of FatA and FatB indicates the N-terminus of the isoforms determines their substrate specificities (Salas J J and Ohlrogge J B (2002) Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases. Arch Biochem Biophys 403(1): 25-34). For those plants which predominantly accumulate medium-chain length saturated fatty acids in seeds, they evolved with specialized FatB and/or FatA thioesterases (Voelker T and Kinney A J (2001) Variations in the biosynthesis of seed-storage lipids. Annu Rev Plant Physiol Plant Mol Biol 52: 335-361). For example, laurate (12:0) is the predominant seed oil in coconut. Correspondingly, the medium-chain specific acyl-ACP thioesterase activity was detected in coconut seeds.

In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and or reduction in the activity of YALI0E16016g (FAT1).

Acyl-CoA Oxidase

Acyl-CoA oxidase (ACO) acts on CoA derivatives of fatty acids with chain lengths from 8 to 18. They are flavoenzymes containing one noncovalently bound FAD per subunit and belong to the same superfamily as mitochondrial acyl-CoA dehydrogenases. Like mitochondrial fatty acyl-CoA dehydrogenases, peroxisomal acyl-CoA oxidases catalyze the initial and rate-determining step of the peroxisomal fatty acid β-oxidation pathway, i.e. α,β-dehydrogenation of acyl-CoA, yielding trans-2-enoyl-CoA in the reductive half-reaction. In the oxidative half-reaction of peroxisomal acyl-CoA oxidase, the reduced FAD is reoxidized by molecular oxygen, producing hydrogen peroxide.

Acyl-CoA oxidase is a homodimer and the polypeptide chain of the subunit is folded into the N-terminal alpha-domain, beta-domain, and C-terminal alpha-domain. Functional differences between the peroxisomal acyl-CoA oxidases and the mitochondrial acyl-CoA dehydrogenases are attributed to structural differences in the FAD environments.

In some embodiments, recombinant microorganisms and methods are provided for the production of short chain fatty alcohols, fatty aldehydes and/or fatty acetates. In certain embodiments, the short chain fatty alcohols, fatty aldehydes and/or fatty acetates have carbon chain length shorter than or equal to C16. In some embodiments, the short chain fatty alcohols, fatty aldehydes and/or fatty acetates are produced from long chain fatty acids. In some preferred embodiments of methods to produce short chain pheromones, select enzymes capable of shortening fatty acyl-CoAs in the pheromone biosynthetic pathway are co-expressed with pheromone biosynthetic pathway enzymes. Examples of suitable chain shortening enzymes include FAD-dependent acyl-CoA oxidase. In the case of fatty acid molecules with an even number of carbons, chain shortening enzymes produce a molecule of acetyl-CoA, and a fatty acyl-CoA shortened by two carbons. Fatty acid molecules with an odd number of carbons are oxidized in a similar fashion producing acetyl-CoA molecules during every round of oxidation until the chain-length is reduced to 5 carbons. In the final cycle of oxidation, this 5-carbon acyl-CoA is oxidized to produce acetyl-CoA and propionyl-CoA.

It is known that acyl-CoA oxidases exhibit varying specificity towards substrates with different chain-length (FIG. 40). Therefore, controlling the degree of fatty acyl-CoA truncation relies on engineering or selecting the appropriate enzyme variant. Examples of acyl-CoA oxidases that are suitable for this purpose are listed in Table 5a.

In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C₄-C₂₂fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA substrate in that cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol. In some embodiments, the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase. In some embodiments, the fatty acyl desaturase has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481. AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the acyl-CoA oxidase is selected from Table 5a. In other embodiments, the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis. Yponometua evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase. In further embodiments, the fatty alcohol forming fatty acyl reductase has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, or 50% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82. In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.

In some preferred embodiments of methods to produce fatty alcohols, fatty aldehydes and/or fatty acetates, one or more genes of the microbial host encoding acyl-CoA oxidases are deleted or down-regulated to eliminate or reduce the truncation of desired fatty acyl-CoAs beyond a desired chain-length. Such deletion or down-regulation targets include but are not limited to Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F0857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g). Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655, CaO19.9224. CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).

In some embodiments, the present disclosure teaches a recombinant microorganism comprising a deletion, disruption, mutation, and or reduction in the activity of one or more acyl-CoA oxidases selected from the group consisting of POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g).

Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions or other disruptions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the proteins encoded by POX1 (YALI0E32835g), Y. lipolytica POX2 (YALI0F10857g). Y. lipolytica POX3 (YALI0D24750g), Y. lipolytica POX4 (YALI0E27654g), Y. lipolytica POX5 (YALI0C23859g), Y. lipolytica POX6 (YALI0E06567g).

Thus, in some embodiments, the recombinant microorganism of the present disclosure will comprise deletions in endogenous genes encoding proteins exhibiting at least 100%, 99%, 98%, 97%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with uniprot database ID POX1(074934), Y. lipolytica POX2 (074935), Y. lipolytica POX3 (074936), Y. lipolytica POX4 (F2Z627), Y. lipolytica POX5 (F2Z630), K. lipolytica POX6 (Q6C6T0).

In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid is provided, wherein the recombinant microorganism expresses one or more acyl-CoA oxidase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyl-CoA oxidase enzymes. In some embodiments, the one or more acyl-CoA oxidase enzymes being expressed are different from the one or more endogenous acyl-CoA oxidase enzymes being deleted or downregulated. In some embodiments, the recombinant microorganism further expresses pheromone biosynthetic pathway enzymes. In further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty acyl desaturase and/or fatty acyl conjugase. In yet further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty alcohol forming fatty acyl reductase. In some embodiments, the one or more acyl-CoA oxidase enzymes that are expressed are selected from Table 5a. In other embodiments, the one or more acyl-CoA oxidase enzymes that are expressed regulate chain length of the mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde and/or fatty acetate. In some embodiments, the one or more endogenous acyl-CoA oxidase enzymes that are deleted, disrupted, mutated, or downregulated are selected from Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W): Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161). In other embodiments, the one or more endogenous acyl-CoA oxidase enzymes that are deleted, disrupted, mutated, or downregulated control chain length of the mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde and/or fatty acetate.

In some embodiments, a method of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid are provided, wherein the method comprises introducing into or expressing in a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyl-CoA oxidase and introducing a deletion, insertion, or loss of function mutation in one or more gene encoding an acyl-CoA oxidase, wherein the at least one endogenous or exogenous nucleic acid molecule encoding an acyl-CoA oxidase being introduced or expressed is different from the one or more gene encoding an acyl-CoA oxidase being deleted or downregulated. In some embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and/or fatty acyl conjugase. In further embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase. In some embodiments, the least one endogenous or exogenous nucleic acid molecule encodes an acyl-CoA oxidase selected from Table 5a. In other embodiments, the least one endogenous or exogenous nucleic acid molecule encodes an acyl-CoA oxidase that regulates chain length of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol, fatty aldehyde and/or fatty acetate. In some embodiments, the one or more gene being deleted or downregulated encodes an acyl-CoA oxidase selected from Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W): Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161). In other embodiments, the one or more gene being deleted or downregulated encodes an acyl-CoA oxidase that regulates chain length of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol, fatty aldehyde and/or fatty acetate.

TABLE 5a

Exemplary acyl-CoA oxidases

Accession No.
Source Organism

P07877

Rama norvegicus

A0A178WDE4

Arabidopsis thaliana

P0CZ23

Arabidopsis thaliana

D7KG20

Arabidopsis lyrata

R0I9Z2

Capsella rubella

V4KEW0

Eutrema salsugineum

M4DG68

Brassica rapa

A0A078IZG1

Brassica napus

A0A087HLF00

Arabis alpina

A0A0D3C825

Brassica oleracea

A0A078FAW4

Brassica napus

A0A178W833

Arabidopsis thaliana

Q9LMI7

Arabidopsis thaliana

D7KG21

Arabidopsis lyrata

A0A0D3C827

Brassica oleracea

M4DG69

Brassica rapa

A0A078J4V6

Brassica napus

A0A078FAY6

Brassica napus

V4KY71

Eutrema salsugineum

A0A061E5C2

Theobroma cacao

A0A061E4K0

Theobroma cacao

M1APJ5

Solanum tuberosum

J7KB16

Prunus persica

K4CXY8

Solanum lycopersicum

V4W234

Citrus clementina

G8XNW7

Malus domestica

F6H4X3

Vitis vinifera

A0A068V5Q3

Coffea canephora

M1APJ6

Solanum tuberosum

A0A067KHP0

Jatropha curcas

A0A0D2PZG8

Gossypium raimondii

B9IQS0

Populus trichocarpa

W9RG01

Morus notabilis

A0A0S3SB10

Vigna angularis var. angularis

A0A0L9V573

Phaseolus angularis

A0A0B0PPT6

Gossypium arboreum

A0A0D2T164

Gossypium raimondii

I1KEV4
Glycine max

I1LS94
Glycine max

G7JUZ2

Medicago truncatula

U5FVP5

Populus trichocarpa

V7AGL5

Phaseolus vulgaris

A0A059A0G8

Eucalyptus grandis

A0A059A0N3

Eucalyptus grandis

A0A166AUM6

Daucus carota subsp. sativus

A0A061EB81

Theobroma cacao

A0A0A0LQY1

Cucumis sativus

A0A022QRB0

Erythranthe guttata

A0A0S3SB01

Vigna angularis var. angularis

A0A0D2Q6S9

Gossypium raimondii

B9SGN6

Ricinus communis

A0A0B2PER8
Glycine soja

A0A0B0NGI2

Gossypium arboreum

A0A0D2SKF2

Gossypium raimondii

A0A0B0NRR7

Gossypium arboreum

A0A0J8EFZ4

Beta vulgaris subsp. vulgaris

A0A0J8BLD2

Beta vulgaris subsp. vulgaris

M4DG71

Brassica rapa

W1Q111

Amborella trichopoda

M0S864

Musa acuminata

A0A166ABS1

Daucus carota subsp. sativus

A0A1D6CA75

Triticum aestivum

A0A0A9CN11

Arundo donax

A0A1D6CKJ3

Triticum aestivum

A0A164W703

Daucus carota subsp. sativus

A0A1D1YDC5

Anthurium amnicola

I1Q2B7

Oryza glaberrima

A0A0D9WQH3

Leersia perrieri

Q69XR7

Oryza sativa subsp. japonica

A0A1D6CA73

Triticum aestivum

A0A0E0A9E1

Oryza glumipatula

A0A199W504

Ananas comosus

A0A0E0HQR9

Oryza nivara

M0T4I4

Musa acuminata subsp. malacc.

C0PTG5

Picea sitchensis

I1I3F1

Brachypodium distachyon

K3XV57

Setaria italica

A0A0D3GGF4

Oryza barthii

A2YCR4

Oryza sativa subsp. indica

A0A0K9RYF5

Spinacia oleracea

A0A0D3GGF3

Oryza barthii

A0A0D3GGF2

Oryza barthii

A0A0E0HQR8

Oryza nivara

A0A1D6CA72

Triticum aestivum

A3BBK8

Oryza sativa subsp. japonica

A0A199V6E4

Ananas comosus

C5XPR4

Sorghum bicolor

A0A0E0PXN1

Oryza rufipogon

B6U7U8

Zea mays

A0A1D6N7A4

Zea mays

A0A0E0E1N7

Oryza meridionalis

A0A0K9NPK9

Zostera marina

A0A059Q1I9

Saccharum hybrid cultivar R570

J3MDZ2

Oryza brachyantha

A0A0K9RYH2

Spinacia oleracea

A0A103YIT3

Cynara cardunculus

A0A0E0PXN2

Otyza rufipogon

A9RZ70

Physcomitrella patens

D8TES8

Selaginella moellendorffii

D8SQF1

Selaginella moellendorffii

M5X7E6

Prunus persica

A9T150

Physcomitrella patens

A0A176WTU5

Marchantia polymorpha

A0A0D2QZ34

Gossypium raimondii

A0A1D6N7A2

Zea mays

A0A1D1Z3C0

Anthurium amnicola

A0A067DSI1

Citrus sinensis

A0A1D6CA74

Triticum aestivum

M8CMI0

Aegilops tauschii

A0A0S3SB72

Vigna angularis var. angularis

M0UX36

Hordeum Vulgare subsp. vulgare

A0A1D6CA76

Triticum aestivum

A0A151SDZ7

Cajanus cajan

Q9LNB8

Arabidopsis thaliana

A0A1D6CKJ4

Triticum aestivum

F2EGJ0

Hordeum vulgare subsp. vulgare

A0A0D2U3V1

Gossypium raimondii

M0UX35

Hordeum vulgare subsp. vulgare

M2Y3U7

Galdieria sulphuraria

S8CGJ3

Genlisea aurea

A0A0E0HQS2

Oryza nivara

A0A199VU62

Ananas comosus

M2WTY9

Galdieria sulphuraria

A0A1D6N7A3

Zea mays

A0A0E0HQS0

Oryza nivara

A0A1E5VL23

Dichanthelium oligosanthes

R7Q711

Chondrus crispus

S0F2R6

Chondrus crispus

S0F2T2

Chondrus crispus

A0A0E0HQS1

Oryza nivara

D3BSZ9

Polysphondylium pallidum

A0A0D2WJ11

Capsaspora owczarzaki strai.

R7QDC3

Chondrus crispus

M1VCW4

Cyanidioschyzon merolae str.

F1A2F0

Dictyostelium purpureum

F4PI57

Dictyostelium fasciculatum

Q54II1

Dictyostelium discoideum

A0A0ELBG6

Oryza punctata

A0A151ZK0

Dictyostelium lacteum

M1BZ65

Solanum tuberosum

H8MFT9

Corralococcus coralloides s.

F8CEB4

Myxococcus fulvus

A0A0H4WJP1

Myxococcus hansupus

Q1CYG7

Myxococcus xanthus

F1A3A8

Dictyostelium purpureum

L7UK64

Myxococcus stipitatus

A0A0F7BPX0

Myxococcus fulvus 124B02

A0A0G4J3N5

Plasmodiophora brassicae

D5H9X3

Salinibacter ruber strain M8

Q2S1W1

Salinibacter ruber strain D.

A0A085WN59

Hyalangium minutum

A0A0G2ZRH9

Archangium gephyra

A0A0S8HAC5

Gemmatimonas sp. SM23_52

A0A177Q5I1

Verrucomicrobia bacterium SC

D8TVM2

Volvox carter f. nagariensis

A0A084SWJ9

Cystobacter violaceus Cb vi76

Q096A6

Stigmatella aurantiaca stra.

R7QMZ0

Chondrus crispus

A0A0G4J5Q9

Plasmodiophora brassicae

A0A0Q9RNC6

Nocardioides sp. Soil797

A0A010GYG34

Cryptosporangium arvum DSM 4

A0A098BJC6

Putative Rhodococcus ruber

A0A059ML28

Rhodococcus aetherivorans

N1M744

Rhodococcus sp. EsD8

W3ZXB8

Rhodococcus rhodochrous ATCC

A0A0A9CKJ6
2 Arundo donax

F4PQH3

Distyostelium fasciculatum

I0Z1P9

Coccomyxa subellipsoidea St.

A0A076ESS0

Rhodococcus opacus

D3BKV2

Polysphondylium pallidum

X0Q4M3

Putative Rhodococcus wratislaviensis

A0A1D6BHN7

Triticum aestivum

C1AZ37

Putative Rhodococcus opacus strain B4

M7ZG40

Triticum urartu

W8HEJ3

Rhodococcus opacus PD630

A0A135GJ74

Rhodococcus sp. SC4

A0A149ZW75

Rhodococcus sp. LB1

Q0SF32

Rhodococcus jostii strain R.

J2JJ09

Rhodococcus sp. JVH1

K8XW36

Rhodococcus opacus M213

A0A152A546

Dictyostelium lacteum

A0A0Q8ZY28

Flavobacterium sp. Root901

L2TJT8

Rhodococcus wratislaviensis

I0WB64

Rhodococcus imtechensis

A0A1B1KC92

Rhodococcus opacus

I3C521

Joostella marina DSM 19592

A0A0F6W8X8

Sandaracimus amylolyticus

I3Z8X9

Belliella baltica strain DS

A0A0J6W3K0

Mycobacterium obuense

A0A0Q7GA13

Flavobacterium sp. Root420

D3BUR8

Polysphondylium pallidum

A0A098SD35

Phaeodactylibcicter xiamenensis

A0A0D1LF86

Mycobacterium llatzerense

A0A0Q5QHB8

Williamsia sp. Leaf354

A0A139VJG5

Mycobacterium phlei DSM 4323

F4PMW9

Dictyostelium fasciculatum

A0A180ERQ3

Lewinella sp. 4G2

Q8MMS1

Dictyostelium discoideum

A0A101CR99

Pavobacteriaceae bacterium

A0A0Q9TDE2

Nocardioides sp. Soil805

A0A0Q9DX23

Flavobacterium sp. Root935

A0A0C1XE41

Hassallia byssoidea VB512170

A0A0J6W7K0

Mycobacterium chubuense

A0A0H4PGA5

Cyclobacterium amurskyense

A0A1B1WLB8

Mycobacterium sp. djl-10

A0A0Q8NET9

Flavobacterium sp. Root186

A0A0J6ZGS8

Mycobacterium chlorophenolicum

A0A085ZIW1

Flavobacterium reichenbachii

J3BZ97

Flavobacteriuin sp. strain C.

A0A066WRY7

Flavobacterium seoulense

K2PYQ1

Galbibacter marinus

A0A0G4IN41

Plasmodiophora brassicae

A0A1B5ZW29

Arenibacter sp. C-21

A0A1B9DW83

Flavobacterium piscis

A0A099CMP1

Mycobacterium rufum

A0A1B2U6C7

Pavobacterium johnsoniae

A0A0M8YPK7

Saccharothrix sp. NRRL B-16348

A0A0T1WAX5

Mycobacterium sp. Root135

K0VAQ4

Mycobacterium vaccae

A0A0Q9JG60

Mycobacterium sp. Soil538

A0A0M0TLE5

Flavobacterium sp. VMW

A0A151ZK81

Dictyostelium lacteum

K6WMK4

Putative Gordonia rhizosphera NBRC 16

L8GZJ4

Acanthamoeba castellanii

I4BLB8

Mycobacterium chubuense

H0RLL0

Putative Gordonia polyisoprenivorans

A0A0C1LEQ5

Prauserella sp. Am3

A0A1E4NXS4

Pseudonocardia sp. SCN 73-27

A0A1A1YGK6

Mycobacterium sp. ACS4331

A0A0G3ILT8

Mycobacterium sp. EPa45

G7GR35

Putative Gordonia amarae NBRC 15530

A0A1A2LUF7

Mycobacterium sp. E136

A0A1A3GTN2

Mycobacterium Mucogenicum

A0A1A0RZ49

Mycobacterium sp. 852002-519..

A0A101AHK0

Mycobacterium sp. IS-1496

A0A126YBZ9

Streptomyces albus

A0A0X3WJ69

Streptomyces griseus subsp.

A0A1C4KUQ6

Streptomyces sp. BvitLS-983

A0A1C4T5K2

Streptomyces sp. OspMP-M43

A0A1A3C0V1

Mycobacterium sp. E740

A0A0G4IKE4

Plosmodiophora brassicae

K1VUE5

Streptomyces sp. SM8

D6B5U8

Streptomyces albus J1074

A0A1C4NBH5

Streptomyces sp. ScaeMP-6W

A0A1C4Q3W7

Streptomyces sp. IgraMP-1

R7WSQ4

Rhodococcus rhodnii LMG 5362

A0A0K2YP95

Rhodococcus sp. RD6.2

XP_011566937

Plutella xylostella peroxisomal

acyl-coenzyme A oxidase 1-like

XP_011568279

Plutella xylostella

probable peroxisomal acyl-coenzyme

A oxidase 1

XP_011568389.1

Plutella xylostella

probable peroxisomal acyl-coenzyme

A oxidase 1

XP_011554180.1

Plutella xylostella

probable peroxisomal acyl-coenzyme

A oxidase 1

XP_011549583.1

Plutella xylostella

probable peroxisomal acyl-coenzyme

A oxidase 3

XP_011548846.1

Plutella xylostella

probable peroxisomal acyl-coenzyme

A oxidase 3

AAP37772
A. thaliana_Acyl-CoA oxidase-3 (ACX3)

XP_011548846.1
Plutella_xylostella_dbmfjv1x1_core_32_

85_1_protein_XP_011548846.1

predicted peroxisomal acyl-coenzyme

A oxidase 3

Acyl Transferases

In some embodiments, recombinant microorganisms and methods are provided for the production of short chain fatty alcohols, fatty aldehydes and/or fatty acetates. In certain embodiments, the short chain fatty alcohols, fatty aldehydes and/or fatty acetates have carbon chain length shorter than or equal to C18. In some preferred embodiments of methods to produce short chain pheromones, select enzymes which prefer to store short-chain fatty acyl-CoAs are co-expressed with one or more fatty acyl desaturase. Such suitable acyltransferase enzymes are exemplified by heterologous or engineered glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs). Examples of acyl transferases that are suitable for this purpose are listed in Table 5b.

In some preferred embodiments of methods to produce fatty alcohols, fatty aldehydes and/or fatty acetates, one or more genes of the microbial host encoding glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs) are deleted or downregulated, and replaced with one or more GPATs, LPAATs, GPLATs, or DGATs which prefer to store short-chain fatty acyl-CoAs. Such deletion or downregulation targets include but are not limited to Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, S. cerevisiae YOR245c, Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881. Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209. In other embodiments, the acyltransferase is inserted at the AXP Acid extracellular protease locus (YALI0B05654g).

Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising an acyltransferase exhibiting at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 700%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 92.

Thus, in some embodiments, the present disclosure teaches a recombinant microorganism comprising a nucleic acid molecule encoding for an acyltransferase, wherein said nucleic acid molecule exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity with any one of SEQ ID Nos. selected from the group consisting of 91.

In some embodiments, the present disclosure teaches a recombinant microorganism comprising at least one nucleic acid molecule encoding an acyltransferase having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% sequence identity to an acyltransferase selected from the group consisting of SEQ ID NO: 92.

Glycerol-3-phosphate acyltransferase (GPAT)

The present disclosure describes enzymes that catalyze the acylation reaction at the sn-1 position of glycerol 3-phosphate shown as follows:

a long-chain acyl-CoA+sn-glycerol 3-phosphate→a 1-acyl-sn-glycerol 3-phosphate+coenzyme A.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation reaction at the sn-1 position of glycerol 3-phosphate. The plant cell contains three types of GPAT, which are located in the chloroplasts, mitochondria and cytoplasm. The enzyme in chloroplasts is soluble and uses acyl-(acyl-carrier protein) as the acyl donor, whereas the enzymes in the mitochondria and the cytoplasm are bound to membranes and use acyl-CoA as the acyl donor (Nishida I et al. (1993) The gene and the RNA for the precursor to the plastid-located glycerol-3-phosphate acyltransferase of Arabidopsis thaliana. Plant Mol Biol. 21(2): 267-77; Murata N and Tasaka Y (1997) Glycerol-3-phosphate acyltransferase in plants. Biochim Biophys Acta. 1348(1-2):10-16).

Eight GPAT genes have been identified in Arabidopsis (Zheng Z et al. (2003) Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility. Plant Cell 15(8):1872-87). GPAT1 was shown to encode a mitochondrial enzyme (Zheng et al. 2003). GPAT4, GPAT5 and GPAT8 were shown to be essential for cutin biosynthesis (Beisson F et al. (2007) The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis. Plant Cell 19(1): 351-368: Li, Y et al. (2007) Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers. Proc Natl Acad Sci USA 104(46): 18339-18344). GPAT2, GPAT3, GPAT6 and GPAT7 have not been characterized yet.

The cytoplasmic GPAT is responsible for the synthesis of triacylglycerol and non-chloroplast membrane phospholipids. It is expected to have a substrate preference for palmitate (C16:0) and oleate (C18:1) since these two fatty acids are the most common ones found at the sn-1 position of plant triacylglycerols. The cytoplasmic GPAT was partially purified from avocado (Eccleston V S and Harwood J L (1995) Solubilisation, partial purification and properties of acyl-CoA; glycerol-3-phosphate acyltransferase from avocado (Persea americana) fruit mesocarp. Biochim Biophys Acta 1257(1): 1-10).

Membrane-bound glycerol-3-phosphate acyltransferase (PlsB) from E. coli catalyzes the first committed step in phospholipid biosynthesis and is thought to function in close proximity to the succeeding enzyme 1-acylglycerol-3-phosphate O-acyltransferase (PlsC) (Kessels J M et al. (1983) Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli. Biochim Biophys Acta 753(2): 227-235). It is specific for acylation at position 1 of sn-glycerol 3-phosphate and can utilize either fatty acyl-acyl carrier protein (acyl-ACP) or fatty acyl-coenzyme A (acyl-CoA) thioesters as acyl donors to form a 1-acyl-sn-glycerol 3-phosphate. Fatty acids that are endogenously synthesized are attached to ACP and exogenously added fatty acids are attached to CoA. In E. coli phospholipids, the sn 1 position is occupied mainly by either palmitate, or cis-vaccenate, whereas the sn 2 position is predominantly palmitoleate, or cis-vaccenate. This is thought to result from the substrate preferences of the PlsB and PlsC enzymes.

The plsB gene has been shown to be regulated by stress response regulators such as RNA polymerase, sigma 24 (sigma E) factor and ppGpp (Wahl A et al. (2011) Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol 80(5): 1260-75. PlsB is part of a protein network for phospholipid synthesis and interacts with a holo-[acyl-carrier protein] (ACP), esterase/thiocsterase (YbgC) and phosphatidylserine synthase (PssA) to form a complex at the cytoplasmic side of the inner membrane.

plsB is essential for growth (Baba T et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2:2006-2008; Yoshimura M et al. (2007) Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli. BMC Microbiol 7: 69).

Site-directed mutagenesis and chemical modification studies have demonstrated catalytically important amino acid residues in PlsB, including an invariant histidine residue that is essential for catalysis (Lewin T M et al. (1999) Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry 38(18): 5764-5771). Genetic studies have identified the plsB locus as involved in the formation of multidrug tolerant persister cells.

The properties of the E. coli B enzyme were studied in earlier work (Kito M et al. (1972) Inhibition of L-glycerol 3-phosphate acyltransferase from Escherichia coli by cis-9,10-methylenehexadecanoic acid. J Biochem 71(1): 99-105; Okuyama H and Wakil S J (1973) Positional specificities of acyl coenzyme A: glycerophosphate and acyl coenzyme A: monoacylglycerophosphate acyltransferases in Escherichia coli. J Biol Chem 248(14): 5197-5205; Kito M et al. (1978) Function of phospholipids on the regulatory properties of solubilized and membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichia coli. Biochim Biophys Acta 529(2): 237-249).

A glycerol-3-phosphate/dihydroxyacetone phosphate dual substrate-specific sn-1 acyltransferase is located in lipid particles and the ER and is involved in the stepwise acylation of glycerol-3-phosphate and dihydroxyacetone in lipid biosynthesis. The most conserved motifs and functionally relevant residues are oriented towards the ER lumen.

A gene (SCT1) encoding a dual glycerol-3-phosphate O-acyltransferase (GAT)/dihydroxyacetone phosphate acyltransferase (DHAT) was identified, cloned and biochemically characterized from Saccharomyces cerevisiae. In the yeast Δgpt1 mutant which exhibits very low GAT/DHAT activity, the overexpression of SCT1 through a plasmid vector showed increased GAT/DHAT activity underlining the proposed molecular function as glycerol-3-phosphate O-acyltransferase/dihydroxyacetone phosphate acyltransferase. The GAT/DHAT activity towards acyl-donors was highest with palmitoleoyl-CoA followed by palmitoyl-CoA, oleoyl-CoA and stearoyl-CoA. The SCT1p was localized to membranes in the cytosol, most probably to the endoplasmic reticulum. In vivo studies of Δsct1 mutants did reveal an impact on all four phospholipids but the observed decrease of 16:0 fatty acids in the phosphatidylethanolamine class was balanced out by an increase in other fatty acids, particularly 18:0 molecular species. The null mutants of SCT1 and GPT2 were synthetically lethal in yeast (Zheng Z and Zou J (2001) The initial step of the glycerolipid pathway: identification of glycerol 3-phosphate/dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae. J Biol Chem 276(45): 417104-41716).

The gene (GPT2) encoding a dual glycerol-3-phosphate O-acyltransferase (GAT)/dihydroxyacetone phosphate acyltransferase (DHAT) from Saccharomyces cerevisiae was identified, cloned and biochemically characterized. GPT2 was recombinantly expressed in E. coli in the ΔplsB background devoid of GAT/DHAT activity and showed an increased GAT activity but could not rescue the mutant probably because of the incorrect embedding of GPT2 in the membrane. In the yeast Δgpt1 mutant which exhibits very low GAT/DHAT activity, the overexpression of GPT2 from a plasmid vector showed increased GAT/DHAT activity, underlining the proposed molecular function as glycerol-3-phosphate O-acyltransferase/dihydroxyacetone phosphate acyltransferase. The GAT/DHAT activity towards acyl-donors was highest with oleoyl-CoA followed by palmitoleoyl-CoA, palmitoyl-CoA and stearoyl-CoA.

The GPT2p was localized to membranes in the cytosol. In vivo studies of Δgpt2 mutants did not reveal any significant impact on the total fatty acid profile but a decrease of 16:1 fatty acids in the phosphatidylethanolamine class was observed which was compensated by an increase in 16:0 and 18:1 molecular species. Analysis of a known yeast mutant TTA1 deficient in GAT activity showed that the TTA1 GPT2 gene had a missense mutation with one nucleotide change in the conserved motif III for acyltransferases. The null mutants of SCT1 and GPT2 were synthetically lethal in yeast (Zheng and Zou 2001).

In some embodiments, the glycerol-3-phosphate acyltransferase is a GPAT from Arabidopsis thaliana (At1g02390). In some embodiments, the glycerol-3-phosphate acyltransferase is PlsB from E. coli (Gene ID EG10740). In some embodiments, the glycerol-3-phosphate acyltransferase is the dual glycerol-3-phosphate O-acyltransferase (GAT)/dihydroxyacetone phosphate acyltransferase (DHAT) SCT1 from S. cerevisiae (YBL011w). In some embodiments, the glycerol-3-phosphate acyltransferase is YALI0C00209g from Yarrowia lipolytica. In some embodiments, the glycerol-3-phosphate acyltransferase is I503_02577 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CTRG_02630 from Candida tropicalis. In some embodiments, the glycerol-3-phosphate acyltransferase is the dual glycerol-3-phosphate O-acyltransferase (GAT)/dihydroxyacetone phosphate acyltransferase (DHAT) GPT2 from S. cerevisiae (YKR067w). In some embodiments, the glycerol-3-phosphate acyltransferase is CaO19.5815 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CaO19.13237 from Candida albicans. In some embodiments, the glycerol-3-phosphate acyltransferase is CTRG_02630 from Candida tropicalis.

Lysophosphatidic Acid Acyltransferase (LPAAT)

The present disclosure describes enzymes that catalyze acylation of the sn-2 position of triacylglycerol.

Membrane-bound I-acylglycerol-3-phosphate O-acyltransferase encoded by gene plsC catalyzes the second step in phospholipid biosynthesis and is thought to function in close proximity to the preceding enzyme glycerol-3-phosphate acyltransferase encoded by gene plsB (Kessels J M et al. 1983). It is specific for acylation at the sn-2 position of a 1-acyl-sn-glycerol 3-phosphate and can utilize either acyl-acyl carrier protein (acyl-ACP), or acyl-coenzyme A (acyl-CoA) as the fatty acyl donor to form a 1,2-diacyl-sn-glycerol 3-phosphate (a phosphatidate, a phosphatidic acid). Fatty acids that are endogenously synthesized are attached to ACP and exogenously added fatty acids are attached to CoA (Greenway D L and Silbert D F (1983) Altered acyltransferase activity in Escherichia coli associated with mutations in acyl coenzyme A synthetase. J Biol Chem 258(21): 13034-13042). In E. coli phospholipids at the sn 1 position is occupied mainly by either palmitate, or cis-vaccenate, whereas the sn 2 position is predominantly palmitoleate, or cis-vaccenate. This is thought to result from the substrate preferences of the PlsB and PlsC enzymes (Rock C O et al. (1981) Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol 3-phosphate. J Biol Chem 256(2): 736-742; Goelz S E and Cronan J E (1980) The positional distribution of fatty acids in Escherichia coli phospholipids is not regulated by sn-glycerol 3-phosphate levels. J Bacteriol 144(1): 462-464).

Site directed mutagenesis studies showed that changing threonine-122 to alanine or leucine resulted in changes in acyl-CoA substrate specificity (Morand L Z et al. (1998) Alteration of the fatty acid substrate specificity of lysophosphatidate acyltransferase by site-directed mutagenesis. Biochem Biophys Res Commun 244(1): 79-84).

In an engineered strain of E. coli, overexpression of PlsC and GalU resulted in increased production of glycoglycerolipids (Mora-Buye N et al. (2012). An engineered E. coli strain for the production of glycoglycerolipids. Metab Eng 14(5): 551-559).

The plsC gene of Streptococcus pneumoniae encodes a 1-acylglycerol-3-phosphate acyltransferase homologous to the E. coli enzyme. The gene was cloned and expressed in E. coli, and membranes expressing it were shown to catalyze the predicted function (Lu Y J et al. (2006) Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens. Mol Cell 23(5): 765-772).

Plant lysophosphatidate acyltransferase (LPAAT) catalyzes acylation of the sn-2 position of triacylglycerol. The substrate specificity of LPAAT in a given plant species generally determines what fatty acid species are incorporated at the sn-2 position. LPAAT has been cloned from maize and meadow foam. There are two LPAAT genes in meadow foam, whereas only one in maize. The enzyme activity of both LAT1 and LAT2 in meadow foam was confirmed by in vitro assay. In addition, LAT2 was shown to functional complement the E. coli LPAAT deficient strain (Brown A P et al. (2002) Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene. Biochem J 364(Pt 3):795-805).

LAT1 is a highly selective acyltransferase that only uses 18:1-CoA as a substrate. LAT2 is less selective. The highest activity was shown towards 22:1-CoA, followed by 16:0- and 18:1-CoA. The substrate specificities of LAT1 and LAT2 are consistent with their proposed roles, for LAT1 in membrane lipid biosynthesis and LAT2 in storage lipid biosynthesis. Plant cell membranes predominantly contain C16 and C18 unsaturated fatty acids, whereas storage lipids contain a wide range of fatty acids including saturated fatty acids and very long-chain unsaturated fatty acids. The protein level of LAT1 and LAT2 in different plant tissues was detected by antibodies. LAT1 is present in both leaves and developing seeds, whereas LAT2 is only detected in developing seeds. This again is consistent with their proposed roles. The role of LAT2 in triacylglycerol biosynthesis was further shown by transformation of LAT2 in oil seed rape which does not normally contain 22:1-CoA at the sn-2 position. The transformation of the meadow foam LAT2 inserted 22:1-CoA at the sn-2 position (Lassner M W et al. (1995) Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil. Plant Physiol 109(4): 1389-1394).

Utilizing viable mutant Saccharomyces cerevisiae strains lacking sphingolipid biosynthesis, the gene SLC1 was isolated and demonstrated to encode an acyl-CoA; lysophosphatidate acyltransferase. Sequence homology with the PLSC protein of E. coli which is classified as 1-acyl-sn-glycerol-3-phosphate acyltransferase indicated a similar function. This presumed molecular function of SLC1p was corroborated by the ability to rescue the ΔplsC mutant of E. coli. It could be shown that a single nucleotide alteration changing an L-glutamine to an L-leucine at position 131 transformed the substrate preference from C16 and C18 fatty acids to C26 fatty acids which was reflected in vivo in the corresponding fatty acid composition of wild type (SLC1) versus mutant (SLC1-1) (Nagiec M M et al. (1993) A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase. J Biol Chem 268(29): 22156-22163).

In vitro assays with the recombinantly expressed and purified SLC1p in E. coli revealed a substrate preference towards lyso-phosphatidate and oleoyl-CoA but also accepted 1-palmitoylglycerol 3-phosphate and 1-stearoyl-sn-glycerol 3-phosphate. In vivo studies of mutants such as Δslc1, Δslc4 (another potential acyl-CoA:phosphatidyl acyltransferase) and double mutants of Δslc1 Δslc4 bearing a plasmid with either the SLC1 or SLC4 gene referred to as 2.ΔSLC1 (or 2.ΔSLC4) showed that SLC1 promoted the biosyntheses of phosphatidate and also phosphatidylinositol and diacylglycerol. It was suggested that SLC1 is involved in phospholipid remodeling by exchanging fatty acids on glycerophospholipids in vive (Benghezal M et al. (2007) SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast. J Biol Chem 282(42): 30845-30855).

Screening the yeast genome with candidate open reading frames (ORFs) of known acyltransferase enzymes and testing the associated deletion strains, the gene encoding an acyl-CoA dependent lyso-phospholipid acyltransferase (ALE1) was identified. In the Δale1 strain a dramatic decrease of lyso-phosphatidylethanolamine acyltransferase (LPEAT) activity was observed but it could also be demonstrated that ALE1p may provide redundant lyso-phosphatidate acyltransferase (LPAAT) activity when the main LPAAT in Saccharomyces cerevisiae, i.e. SLC1p, is absent or rendered inactive. ALE1p preferably attaches unsaturated acyl chains of varying length to the sn-2 position of lyso-phospholipids. The enzyme was localized to both microsomal and mitochondrial membranes utilizing high purity cell fractionation. It has been proposed that ALE1 may be the major LPEAT in the exogenous lysolipid metabolism (ELM) pathway in yeast but it is also required for efficient functioning of the endogenous Kennedy pathway (Riekhof W R et al. (2007) Identification and characterization of the major lysophosphatidylethanolamine acyltransferase in Saccharomyces cerevisiae. J Biol Chem 282(39): 28344-28352).

In a simultaneous study, LPT1 (synonymous to ALE1) was identified by applying a synthetic genetic array analysis and shown to have lyso-phospholipid acyltransferase activity. In this study the best substrate for LPT1 (=ALE1) was lyso-phosphatidylcholine, hence acting as a lyso-phosphatidylcholine acyltransferase (LPCAT) and the residual activity as LPAAT reported earlier was also demonstrated utilizing single Δlpt1 and double Δscl1Δlpt1 mutants, the latter being inviable. The ratio of incorporating oleate into phosphatidylcholine was determined as 70% towards the de novo synthesis and 30% towards remodeling (Jain S et al. (2007) Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae. J Biol Chem 282(42): 30562-30569).

The molecular function of ALE1 (also referred to as LCA1 or SLC4) as a lyso-phosphatidylcholine acyltransferase (LPCAT) was corroborated in another simultaneous study monitoring the incorporation of radioactive labeled lyso-phosphatidylcholine and/or palmitoyl-CoA into phosphatidylcholine (PC). The study confirmed that ALE1p (=LCA1p in this study) was accepting a variety of acyl-donors but showed highest activity as LPCAT regardless of the acyl-chain of lyso-phosphatidylcholine species (16:0 or 18:1). In addition, a high sensitivity towards Zn2+ was observed which was inhibitory at concentrations above 0.1 mM and activating at lower concentrations (10 to 25 μM). The high PC turnover-rate measured for ALE1p (=LCA1p) emphasized the enzyme as a key catalyst involved in the re-acylation of PC (Chen Q et al. (2007) The yeast acylglycerol acyltransferase LCA1 is a key component of Lands cycle for phosphatidylcholine turnover.” FEBS Lett 581(28): 5511-5516).

The search for genes causing aberrations in the formation of lipid droplets (LD) in Saccharomyces cerevisiae identified the gene LOA1 (formerly VPS66) encoding for an acyl-CoA dependent lysophosphatidate acyltransferase. The in vivo molecular function of LOA1p was determined using the comparison of the lipidome of wild type and Δloa1 yeast strains. The analysis showed that in the LOA1 deficient mutant (Δloa1) the percentage of oleate containing phosphatidate molecular species was considerably reduced and the content of triacylglycerols (TGA) was lowered by 20 percent. The protein was recombinantly expressed in E. coli and partially purified by obtaining the highly enriched lipid droplet fraction and by affinity chromatography with LOA1p still attached to the matrix beads. The purified LOA1p was characterized in in vitro assays demonstrating that LOA1p was specific for lysophosphatidate and oleoyl-CoA, thus acting as a oleoyl-CoA; lysophosphatidate acyltransferase in yeast. Based upon the results, LOA1p was proposed to be significantly involved in channeling excess oleate—containing phosphatidate species into TAG biosynthesis and the proper development of lipid droplets (LD's). Utilizing a genomic-tagging construct, subcellular fractionation, immunohistochemistry and fluorescence microscopy LOA1 could be localized to both endoplasmic reticulum (ER) and lipid droplets (LD's) (Ayciriex S et al. (2012) YPR139c/LOA encodes a novel lysophosphatidic acid acyltransferase associated with lipid droplets and involved in TAG homeostasis. Mol Biol Cell 23(2): 233-246).

In some embodiments, the lysophosphatidic acid acyltransferase is plsC from E. coli (MetaCyc Accession ID EG11377). In other embodiments, the lysophosphatidic acid acyltransferase is plsC from S. pneumoniae (MetaCyc Accession ID G-10763). In some embodiments, the lysophosphatidic acid acyltransferase is LAT1 from Limnanthes douglasii. In some embodiments, the lysophosphatidic acid acyltransferase is LAT2 from Limnanthes douglasii (MetaCyc Accession ID G-9398). In some embodiments, the lysophosphatidic acid acyltransferase is SLC1 from Saccharomyces cerevisiae (YDL052c). In some embodiments, the lysophosphatidic acid acyltransferase is YALI0E18964g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.250 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.7881 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRG_02437 from Candida tropicalis. In some embodiments, the lysophosphatidic acid acyltransferase is ALE1 from Saccharomyces cerevisiae (YOR175C). In some embodiments, the lysophosphatidic acid acyltransferase is YALI0F19514g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.1881 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.9437 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRG_01687 from Candida tropicalis. In some embodiments, the lysophosphatidic acid acyltransferase is LOA1 from Saccharomyces cerevisiae (YPR139C). In some embodiments, the lysophosphatidic acid acyltransferase is YALI0C14014g from Yarrowia lipolytica. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.1043 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CaO19.8645 from Candida albicans. In some embodiments, the lysophosphatidic acid acyltransferase is CTRG_04750 from Candida tropicalis.

Glycerolphospholipid Acyltranserase (GPLAT)

The present disclosure describes enzymes that catalyze the following reaction:

1-alkyl-sn-glycero-3-phosphoethanolamine+a 2-acyl-1-alkyl-sn-glycero-3-phosphocholine≈an O-1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine+a 1-alkyl-2-lyso-sn-glycero-3-phosphocholine

GPLAT enzymes catalyze the transfer of fatty acids from intact choline- or ethanolamine-containing glycerolphospholipids to the sn-2 position of a lyso-glycerolphospholipid. The organyl group on sn-1 of the donor or acceptor molecule can be alkyl, acyl or alk-1-enyl. The term ‘radyl’ has sometimes been used to refer to such substituting groups. The enzyme requires Coenzyme A and does not favor the transfer of polyunsaturated acyl groups.

Diacylglycerol Acyltransferase (DGAT)

The present disclosure describes enzymes that add an acyl group to the sn-3 position of diacylglycerol (DAG) to form triacylglycerol (TAG).

Diacylglycerol acyltransferase (DGAT) catalyzes the only unique reaction in triacylglycerol biosynthesis. It adds an acyl group to the sn-3 position of diacylglycerol (DAG) and forms triacylglycerol (TAG), shown as follows:

an acyl-CoA+a 1,2-diacyl-sn-glycerol→a triacyl-sn-glycerol+coenzyme A.

DGAT accepts a broad range of acyl-CoA as acyl donor including C18:1, C18:2, and C20:1 acyl-CoA as demonstrated for the Arabidopsis DGAT (Jako C et al. (2001) Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol 126(2): 861-874). Expressing the Arabidopsis cDNA of DGAT in an insect cell culture and in yeast, as well as over-expressing the cDNA in wild type Arabidopsis, demonstrated the DGAT activity in transferring an acyl group to the sn-3 position of DAG (Hobbs D H et al. (1999) Cloning of a cDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression. FEBS Lett 452(3): 145-149; Zou J et al. (1999) The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19(6): 645-653). Over-expression of the Arabidopsis cDNA in wild type Arabidopsis increased oil deposition in seeds and this increase is correlated to the increased mRNA expression level of DGAT. This indicates that DGAT is a regulatory point of the triacylglycerol biosynthesis pathway.

The gene encoding the bifunctional acyl-CoA:acylglycerol acyltransferase (DGAT) has been identified in Saccharomyces cerevisiae as a major contributor to triacylglycerol biosynthesis (Sandager L et al. (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277(8): 6478-6482). The gene (DGA1) belongs in the DGAT2 family which members are characterized as acyl-CoA dependent acyltransferases (Lardizabal K D et al. (2001) DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity.” J Biol Chem 276(42): 38862-38869). It has been demonstrated that DGA1p is the only acyl-CoA dependent acyltransferase catalyzing the esterification of diacylglycerol (DAG) to triacylglycerol (TAG) in the yeast genome. This has been shown in deletion mutants of DGA1 (Δdga1) and in combination with the deletion of the other diacylglycerol acyltransferase of importance in yeast, i.e. LRO1 which esterifies DAG utilizing a phospholipid acyl donor (Δlro1). In the Δdga1Δlro1 double mutant almost all of the diacylglycerol acyltransferase has been lost and TAG synthesis was abolished. A plasmid carrying the DGA1 gene could rescue the TAG synthetic deficiency in the mutant indicating that in vivo DGA1 was prominently involved in the TAG biosynthetic route (Sorger D, Daum G (2002). Synthesis of triacylglycerols by the acyl-coenzyme A:diacylglycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces cerevisiae. J Bacteriol 184(2): 519-524: Oelkers P et al. (2002) The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem 277(11): 8877-8881). In vitro a preference of DGA1p towards oleoyl-CoA and palmitoyl-CoA was observed which is inverted for the phospholipid dependent acyltransferase LRO1p (Oelkers et al. 2002).

In addition, the function of DGA1p as an acyl-CoA dependent monoacylglycerol acyltransferase (MGAT) was demonstrated in vivo utilizing Δdga1 mutants which had lost more than 60% of the MGAT activity. The in vitro MGAT activity of DGA1 was shown by the oleoyl-CoA dependent esterification of 2-oleoylglycerol yielding 1,2-dioleoylglycerol in the process (Heier C et al. (2010) Identification of Yju3p as functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1801(9): 1063-1071).

More insights into the functional importance and topological orientation of sequence motifs in the primary sequence of DGA1p has been gained by in silico analyses, site-directed mutagenesis of signature motifs and deletion mutations of the C- and N-termini. It could be demonstrated that besides the signature motifs found in other DGAT2 family members Saccharomyces possesses a unique hydrophilic stretch which was shown to significantly modulate enzyme activity. Also, the histidine residue 195 in the second of the four determined transmembrane domains was proven to be essential for enzyme activity. The topology of DGA1 revealed that both C- and N-termini face the cytoplasm and that the C-terminus was more important for DGA1 activity than the N-terminus (Liu Q et al. (2011) Functional and topological analysis of yeast acyl-CoA:diacylglycerol acyltransferase 2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis. J Biol Chem 286(15): 13115-13126).

Using highly purified cell fragments and immunoblotting, Sorger et al. (2002) and Liu et al. (2011) demonstrated that DGA1 was localized to lipid droplets and microsomal membranes, most probably the endoplasmic reticulum.

Acinetobacter sp. ADP1 expresses a bifunctional enzyme that exhibits both wax ester synthase (WS) and acyl-coA:diacylglycerol acyltransferase (DGAT) activities (Kalscheuer R and Steinbuchel A (2003) A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278(10): 8075-8082). This homodimer catalyzes the final steps in TAG and WE biosynthesis (Stoveken T et al. (2005) The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 187(4): 1369-1376). It mediates both oxo ester and thio ester bond formation and has a broad substrate range, accepting medium chain fatty alcohols and acyl-CoA esters as well as monoacylglycerols (MAGs) (Uthoff S et al. (2005) Thio wax ester biosynthesis utilizing the unspecific bifunctional wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase of Acinetobacter sp. strain ADP1. Appl Environ Microbiol 71(2): 790-796).

In some embodiments, the diacylglycerol acyltransferase is TAG1 from Arabidopsis thaliana (Gene ID AT2G19450). In some embodiments, the diacylglycerol acyltransferase is DGA1 from S. cerevisiae (YOR245c). In some embodiments, the diacylglycerol acyltransferase is atfA from Acinetobacter sp. ADP1 (MetaCyc Accession ID ACIAD0832). In some embodiments, the diacylglycerol acyltransferase is YALI0E32769g from Yarrowia lipolytica. In some embodiments, the diacyiglycerol acyltransferase is CaO19.6941 from Candida albicans. In some embodiments, the diacylglycerol acyltransferase is CaO19.14203 from Candida albicans. In some embodiments, the diacylglycerol acyltransferase is CTRG_06209 from Candida tropicalis.

Phospholipid: diacylglycerol acyltransferase (PDAT) catalyzes the following reaction:

a phosphatidylcholine+a 1,2-diacyl-sn-glycerol→a triacyl-sn-glycerol+a 1-acyl-sn-glycero-3-phosphocholine.

The Arabidopsis PDAT can use different phospholipids as acyl donor, with acyl groups of 10-22 carbon chain length at either sn-positions (Stahl U et al. (2004) Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis. Plant Physiol 135(3):1324-1335). Acyl group at the sn-2 position of phosphatidylcholine is however used three times greater than at the sn-1 position. The highest activity is with acyl groups having multiple double bonds, epoxy or hydroxy groups. Among the tested, the enzyme activity was highest with ricinoleoyl, 18:0- and 22:1-acyl groups gave the lowest enzyme activity. Among different phospholipid species, higher activity is with phosphatidylethanolamine than with phosphatidate or phosphatidylcholine.

A PDAT activity was detected in castor bean seed microsome fraction. Radio-labeled ricinoleoyl and vernoloyl groups are effectively transferred from phosphatidylcholine to DAG forming triacylglycerol (Dahlqvist A et al. (2000) Phospholipid: diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci USA 97(12): 6487-6492).

In other embodiments, the diacylglycerol acyltransferase is a phospholipid: diacylglycerol acyltransferase (PDAT). In some embodiments, the PDAT is from Arabidopsis thaliana (Gene ID AT5G13640). In some embodiments, the PDAT is from Ricinus communis. In some embodiments, the PDAT is LRO1 from Saccharomyces cerevisiae (YNR008w). In some embodiments, the PDAT is YALI0E6797g from Yarrowia lipolytica. In some embodiments, the PDAT is CaO19.13439 from Candida albicans. In some embodiments, the PDAT is CTRG_04390 from Candida tropicalis.

In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid is provided, wherein the recombinant microorganism expresses one or more acyltransferase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyltransferase enzymes. In some embodiments, the one or more acyltransferase enzymes being expressed are different from the one or more endogenous acyltransferase enzymes being deleted or downregulated. In some embodiments, the one or more endogenous or exogenous acyltransferase enzymes comprise glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs). In some embodiments, the one or more acyltransferase enzymes being expressed prefer to store short-chain fatty acyl-CoAs. In other embodiments, the one or more acyltransferase enzymes being expressed are selected from Table 5b. In some embodiments, the one or more endogenous acyltransferase enzymes being deleted or downregulated are selected from Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, S. cerevisiae YOR245c, Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209. In some embodiments, the recombinant microorganism further expresses pheromone biosynthetic pathway enzymes. In further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty acyl desaturase and/or fatty acyl conjugase. In yet further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty alcohol forming fatty acyl reductase.

In some embodiments, a method of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid are provided, wherein the method comprises introducing into or expressing in a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase and introducing a deletion, insertion, or loss of function mutation in one or more gene encoding an acyltransferase, wherein the at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase being introduced or expressed is different from the one or more gene encoding an acyltransferase being deleted or downregulated. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase being introduced or expressed or the one or more gene encoding an acyltransferase being deleted or downregulated comprise glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs). In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encodes an acyltransferase that prefers to store short-chain fatty acyl-CoAs. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encodes an acyltransferase selected from Table 5b. In some embodiments, the one or more endogenous acyltransferase enzymes being deleted or downregulated are selected from Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C. S. cerevisiae YPR139C, S. cerevisiae YNR008w, S. cerevisiae YOR245c, Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437. Candida CTRG_01687, Candida CaO19.1043. Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390. Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209. In some embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and/or fatty acyl conjugase. In further embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase.

TABLE 5B

Exemplary acyltransferases

Accession No.
Source Organism

AALA9962.1

Bos taurus

BAC43739.1

Rattus norvegicus

AAH89846.1

Rattus norvegicus

F6TMU0

Equus caballus

F6PXX7

Equus caballus

F7B020

Equus caballus

ALT83519.1

Macadamia tetraphylla

ANN46862.1

Cuphea avigera

ANN46863.1

Cuphea avigera

ANN46864.1

Cuphea avigera

ANN46865.1

Cuphea avigera

AAC49119.1

Cocos mucifera

JAT48335.1

Anthurium amnicola

XP_008793203.1

Phoenix dactylifera

XP_008806896.1

Phoenix dactylifera

XP_008806740.1

Phoenix dactylifera

XP_010908895.1

Elaeis guineensis

XP_010908896.1

Elaeis guineensis

Q96UY2

Umbelopsis ramanniana

A0A077WEU5

Lichtheimia ramosa

A0A068SDP4

Lichtheimia corymbifera JMRC

A0A068RXA2

Lichtheimia corymbifera JMRC

A0A197JCE2

Mortierella elongata AG-77

A0A1C7N060

Choanephora cucurbitarum

I1BLC3

Rhizopus delemar

A0A1C7NC56

Choanephora cucurbitarum

A0A077X3B5

Lichtheimia ramosa

Q96UY1

Umbelopsis ramanniana

A0A077WVD4

Lichtheimia ramosa

A0A163K8G3

Absidia glauca

S2J8P3

Mucor circinelioides

A0A168J818

Mucor circinelloides

A0A0C9MR10

Mucor ambiguus

A0A162PN39

Phycomyces blakesleeanus

A0A167QXD0

Phycomyces blakesleeanus

A0A0C9M4C3

Mucor ambiguus

A0A0R7NDT1

Parasitella parasitica

A0A015LM78

Rhizophagus irregularis

A0A0B7NHQ3

Parasitella parasitica

A0A0A1NVK5

Rhizopus microsporus

A0A0A1P436

Rhizopus microsporus

A0A0D7BI48

Cylindrobasidium torrendii

A0A1B9HZT8

Kwoniella pini

A0A1D1XN50

Anthurium amnicola

A0A1B9ILF0

Kwoniella mangroviensis

S2JU94

Mucor circineiloides

A0A1B9GCB0

Kwoniella bestiolae CBS 10118

A0A068RKT0

Lichtheimia corymbifera

Q5KFU4

Cryptococcus neoformans

Q55QC2

Cryptococcus neoformans

U5GY58

Microbotryum lychnidis

A0A197KA94

Mortierella elongata AG-77

A0A088FR92

Rhodotorula diobovata

A0A194SBY3

Rhodotorula graminis

E6R8N8

Cryptococcus gattii

M7WKS9

Rhodosporidium toruloides

A0A191UMW0

Rhodosporidium toruloides

C6KZS6

Rhodosporidium toruloides

J9VS50

Cryptococcus neoformans

A0A109FM23

Rhodotorula sp. JG-1b

I4YE91

Wallemia mellicola

A0A066WAJ3

Tilletiaria anomala UBC 951

A0A151VHJ4

Hypsizygus marmoreus

A0A168LDJ3

Absidia glauca

A0A0A1ULK8

Rhizoctonia solani AG-3 Rhs1AP

A0A074RWU7

Rhizoctonia solani 123E

A0A0K6FWT6

Rhizoctonia solani

R9AL76

Wallemia ichthyophaga

E6ZMU5

Sporisorium reilianum

A0A0K3CJX4

Rhodosporidium toruloides

A0A162Y103

Phycomyces blakesleeanus

A0A0B7FYU9

Thanatephorus cucumeris

A0A1A5ZUI2

Kwoniella dejecticola

A0A1B9GXE9

Kwoniella heveanensis BCC8398

V5EIP7

Kalmanozyma brasiliensis

A0A127ZHG0

Sporisorium scitamineum

M5FTN9

Dacryopinax primogenitus

A0A166HX72

Sistotremastrum suecicun

A0A067QH80

Jaapia argillacea MUCL 33604

A0A165PFB6

Neolentinus lepideus

G7DXE4

Mixia osmundae

A0A165KJK5

Exidia glandulosa HHB12029

A0A0F7TLQ7

Penicillium brasilianum

S8FI87

Fomitopsis pinicola

S7ZL04

Penicillium oxalicum

I2FMX3

Ustilago hordei

F8P370

Serpula lacrymans

V2WTH2

Moniliophthora roreri

S7Q9H4

Gloeophyllum trabeum

W3VTZ4

Pseudozyma aphidis

B8M0V7

Talaromyces stipitatus

A0A0D7B6H5

Cylindrobasidium torrendii

R7SCW4

Tremella mesenterica

A0A093UWD0

Talaromyces marneffei PM1

B6Q8Q9

Talaromyces marneffei

A0A093VC12

Talaromyces marneffei PM1

A0A167SF58

Calocera viscosa TUFC12733

A0A180GQ68

Puccinia triticina

E3KWZ5

Puccinia graminis f. sp.

F4S978

Melampsora larici-populina

A0A0U5GN87

Aspergillus calidoustus

W9WBT1

Clacdophialophora yegresii

A0A0D2A9G0

Verruconis gallopava

S3DKQ1

Glarea lozoyensis

A0A167S691

Penicillin-in chrysogenum

A0A0C3G1P8

Piloderma croceum F 1598

A0A117NM34

Penicillium freii

A0A0M8NPT1

Penicillium nordicum

M2R3J5

Ceriporiopsis subvermispora

A0A1E3JS60

Cryptococcus depauperatus

V9DJY4

Cladophialophora carrionii

A0A1C1D128

Cladophialophora carrionii

A0A194XRZ1

Phialocephala scopiformis

A0A135LQY4

Penicillium patulum

F2S034

Trichophyton tonsurans

A0A059J710

Trichophyton interdigitale

R7YTC1

Coniosporium apollinis

A0A0G4PR11

Penicillium camemberti FM 013

F2SHG6

Trichophyton rubrum

A0A022VWY8

Trichophyton rubrum CBS 288.86

A0A178F1Q9

Trichophyton rubrum

A0A022XM67

Trichophyton soudanense

F2PHM1

Trichophyton equinum

A0A178FDV0

Trichophyton violaceum

A0A0F8UUV5

Aspergillus ochraceoroseus

A0A0F8XD12

Aspergillus rambellii

D8Q1Z6

Schizophyllum commune

A0A0L0VQ99

Puccinia striiformis

W6QE33

Penicillium roqueforti

A0A0J0XU39

Cutaneotrichosporon

K2RIY7

Macrophomina phaseolina

A0A1B9HIE8

Kwoniella heveanensis CBS 569

A0A0A2KLE4

Penicillium italicum

A0A177FP94

Fonsecaea monophora

Q0CU51

Aspergillus terreus

A0A0D2C195

Exophiala spinifera

K9GS70

Penicillium digitatum

K9H4T7

Penicillium digitatum

A0A0A2IRX2

Penicillium expansum

A0A165XA55

Fibulorhizoctonia sp.

A0A1E3HS30

Cryptococcus depauperatus

R0JHT6

Setosphaeria turcica

W6XT38

Bipolaris zeicola 26-R-13

K1WNS8

Marssonina brunnea f. sp.

A0A077R6Q5

Melanopsichium pennsylvanicum

A0A0G2F2K4

Phaeomoniella chlamydospora

M2UB23

Cochliobolus heterostrophus

N4WZB4

Cochliobolus heterostrophus

A0A0D2ECJ4

Capronia semi-immersa

K5ULK6

Phanerochaete carnosa

A0A081CNS6

Pseudozyma Antarctica

W7E3D1

Bipolaris victoriae FI3

A0A0D1YAT0

Exophiala sideris

V5FVB4

Byssochlamys spectabilis

A0A150V2J4

Acidomyces richmondensis BFW

A0A0D2P224

Hvpholoma sublateritium

C5FY83

Arthroderma otae

A0A0E9NND3

Saitoella complicata

A0A163JYI7

Absidia glauca

M2SYN8

Cochliobolus sativus

A0A0D2A9Y8

Exophiala oligosperma

B2WFQ5

Pyrenophora tritici

A0A178Z686

Fonsecaea erecta

R1GYF1

Botryosphaeria parva

A0A0D2AM77

Cladophialophora immunda

A0A067TPJ7

Galerina marginata CBS 339.88

A0A0G2DT71

Diplodia seriata

A0A0S6XG57
fungal sp. No. 11243

A1CD57

Aspergillus clavatus strain

W6ZE59

Bipolaris oryzae ATCC 44560

W9X299

Cladophialophora psammophila

A0A0L1HS74

Stemphylium lycopersici

E3RYE6

Pyrenophora teres

A0A178C491

Fonsecaea multimorphosa

A0A0D2JW30

Fonsecaea multimorphosa

A0A100ISZ7

Aspergillus niger

G7XRR4

Aspergillus kawachii

E4ZGH1

Leptosphaeria maculans

A0A0C3AU69

Serendipita vermifera

A0A0U1M481

Talaromyces islandicus

A0A179UDB8

Ajellomyces dermatitidis

A0A177DML0

Alternaria alternata

A0A074XTA2

Aureobasidium namibiae

R8BK00

Togninia minima

A0A178E1M9

Pyrenochaeta sp. DS3sAY3a

A0A074XCF2

Aureobasidium pullulans

A0A178CVL7

Fonsecaea nubica

J4R349

Fibroporia radiculosa

F2T2H3

Ajellomyces dermatitidis

T5C9R0

Blastomyces dermatitidis

C5GGF5

Ajellomyces dermatitidis

F8Q4F5

Serpula lacrymans

A0A074YHW3

Aureobasidium subglaciale

A0A0D2E953

Exophiala xenobiotica

A0A0D2ETM7

Exophiala xenobiotica

A0A163ADJ9

Didymella rabiei

U7PLY5

Sporothrix schenckii

A0A0F2MF45

Sporothrix schenckii 1099-18

A0A0C2J820

Sporothrix brasiliensis 5110

A0A1E3B843

Aspergillus cristatus

A0A01L6WTD3

Termitomyces sp. J132

G2YTS7

Botryotinia fuckeliana

W9XGA9

Capronia epimyces CBS 606.96

A0A0F4YS69

Rasamsonia emersonii

M9LWR9

Pseudozyma antarctica

A0A074WDM7

Aureobasidium melanogenum

M3CBZ0

Sphaerulina musiva

A0A0C7C2J7

Rhizopus microsporus

W9YU83

Capronia coronata CBS 617.96

I8IUH8

Aspergillus oryzae

A0A139HZI0

Pseudocercospora musae

E9DGY4

Coccidioides posadasii

A0A0J6F9P8

Coccidioides posadasii

H6BM52

Exophiala dermatitidis

Q2UDX3

Aspergillus oryzae

M3ASJ4

Pseudocercospora fijiensis

A0A177BZU0

Paraphaeosphaeria sporulosa

A0A017S910

Aspergillus ruber CBS 135680

A0A175VVF2

Madurella mycetomatis

A0A0J8UWI6

Coccidioides immitis

A0A0J6YFS7

Coccidioides immitis RMSCC

J3K3F7

Coccidioides immitis

A0A0D2FX82

Rhinocladiella mackenziei

A0A072PSS5

Exophiala aquamarina

A0A0A1MWE2

Rhizopus microsporus

W2RSU8

cyphellophora europaea

C0S1D5

Paracoccidioides brasiliensiensis

C1G9R2

Paracoccidioides brasiliensiensis

A0A1D2JGH6

Paracoccidioides brasiliensis

A0A166PXN0

Cordyceps brongniartii

Q54GC1

Dictyostelium discoideum

A0A0H1B9A9

Emmonsia parva UAMH 139

R4XEF3

Taphrina deformans

D3B2U8

Polysphondylium pallidum

U1HHT8

Endocarpon pusillum

A0A1E3JYY5

Tsuchiyaea wingfieldii

A0A0C3JN41

Pisolithus tinctorius Marx 270

B6HF05

Penicillium rubens

A0A060S368

Pycnoporus cinnabarinus

K5W449

Agaricus bisporus

B0CTA0

Laccaria bicolor

F0XD96

Grosmannia clavigera

A0A165EP91

Calocera cornea HHB12733

Acylglycerol Lipases and Sterol Esterases

In some preferred embodiments of methods to produce fatty alcohols, fatty aldehydes and/or fatty acetates, one or more genes of the microbial host encoding acylglycerol lipases (mono-, di-, or triacylglycerol lipases) and sterol ester esterases are deleted or downregulated and replaced with one or more acylglycerol lipases which prefer long chain acylglycerol substrates. Such deletion or down-regulation targets include, but are not limited to Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, S. cerevisiae YLR020C, Candida CaO19.2050, Candida CaO19.9598. Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426. Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.

Carboxylic ester hydrolases (EC 3.1.1) are a large class of enzymes catalyzing the hydrolysis or synthesis of ester bonds. They have been described in all life domains, prokaryotic and eukaryotic. Most of them belong to the α/β-hydrolase superfamily and have a conserved “catalytic triad” formed by His, an acidic amino acid and a Ser residue that is located in a highly conserved GXSXG sequence. During hydrolysis, the catalytic Ser will start the nucleophilic attack of the substrate helped by the other two residues from the triad, which are in close spatial vicinity. These are presumed to facilitate the hydrolysis of esters by a mechanism similar to that of chymotrypsin-like serine proteases. Another characteristic feature is the presence of an amino acidic region whose sequence is not as conserved as that of the catalytic triad, the oxyanion hole, which serves to stabilize a transition state generated during catalysis. In addition, these enzymes generally do not require cofactors. Acylglycerol lipases and sterol esterases belong to the carboxylic ester hydrolase family.

An acylglycerol lipase enzyme catalyzes a chemical reaction that uses water molecules to break the glycerol monoesters of long-chain fatty acids. The systematic name of this enzyme class is glycerol-ester acylhydrolase. Other names in common use include monoacylglycerol lipase, monoacylglycerolipase, monoglyceride lipase, monoglyceride hydrolase, fatty acyl monoester lipase, monoacylglycerol hydrolase, monoglyceridyllipase, and monoglyceridase. This enzyme participates in glycerolipid metabolism.

A sterol esterase enzyme catalyzes the chemical reaction:

steryl ester+H2O⇄sterol+fatty acid

Thus, the two substrates of this enzyme are steryl ester and H2O, whereas its two products are sterol and fatty acid.

The systematic name of this enzyme class is steryl-ester acylhydrolase. Other names in common use include cholesterol esterase, cholesteryl ester synthase, triterpenol esterase, cholesteryl esterase, cholesteryl ester hydrolase, sterol ester hydrolase, cholesterol ester hydrolase, cholesterase, and acylcholesterol lipase. This enzyme participates in bile acid biosynthesis. Sterol esterases are widespread in nature and have been identified from mammals' tissues such as the pancreas, intestinal mucosa, liver, placenta, aorta, and brain, to filamentous fungi, yeast, and bacteria.

In terms of substrate specificity, many sterol esterases are able to catalyze the hydrolysis or synthesis of a rather broad range of other substrates containing ester linkages, such as acylglycerols, aryl esters, and in some cases alcohol esters, cinnamyl esters, xhantophyl esters, or synthetic polymers.

In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid is provided, wherein the recombinant microorganism expresses one or more acylglycerol lipase and/or sterol ester esterase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes. In some embodiments, the one or more acylglycerol lipase and/or sterol ester esterase enzymes being expressed are different from the one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes being deleted or downregulated. In some embodiments, the one or more endogenous or exogenous acylglycerol lipase and/or sterol ester esterase enzymes being expressed prefer to hydrolyze ester bonds of long-chain acylglycerols. In other embodiments, the one or more acylglycerol lipase and/or sterol ester esterase enzymes being expressed are selected from Table 5c. In some embodiments, the one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes being deleted or downregulated are selected from Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, S. cerevisiae YLR020C, Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426. Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630. In some embodiments, the recombinant microorganism further expresses pheromone biosynthetic pathway enzymes. In further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty acyl desaturase and/or fatty acyl conjugase. In yet further embodiments, the pheromone biosynthetic pathway enzymes comprise one or more fatty alcohol forming fatty acyl reductase.

In some embodiments, a method of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acyl-CoA are provided, wherein the method comprises introducing into or expressing in a recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase and introducing a deletion, insertion, or loss of function mutation in one or more gene encoding an acylglycerol lipase or sterol ester esterase, wherein the at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase being introduced or expressed is different from the one or more gene encoding an acylglycerol lipase or sterol ester esterase being deleted or downregulated. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase being introduced or expressed prefers to hydrolyze ester bonds of long-chain acylglycerols. In some embodiments, the at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase or sterol ester esterase being introduced or expressed is selected from Table 5c. In some embodiments, the one or more gene being deleted or downregulated encodes an acylglycerol lipase or sterol ester esterase selected from Y. lipolytica YALI0E32035g. Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C4520g. Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, S. cerevisiae YLR020C, Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185. Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630. In some embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty acyl desaturase and/or fatty acyl conjugase. In further embodiments, the method further comprises introducing into or expressing in the recombinant microorganism at least one endogenous or exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase.

TABLE 5c

Exemplary acylglycerol lipases

Accession No.
Source Organism

EAY76846.1

Oryza sativa

OEL29276.1

Dichanthelium oligosanthes

ONM35522.1

Zea mays

AFW56472.1

Zea mays

AFW60230.1

Zea mays

ACG33769.1

Zea mays

ACG30143.1

Zea mays

ACG39100.1

Zea mays

ACG48810.1

Zea mays

KQK11040.1

Brachypodium distachyon

CAA64004.1

Saccharomyces cerevisiae

CAA81640.1

Saccharomyces cerevisiae

CAG78037.1

Yarrowia lipolytica

EEF47288.1

Ricinus communis

EEF45491.1

Ricinus communis

EEF52390.1

Ricinus communis

EEF38788.1

Ricinus communis

EER38789.1

Ricinus communis

EEF28563.1

Ricinus communis

EEF46013.1

Ricinus communis

AFQ93681.1

Ricinus commums

EEF45592.1

Ricinus communis

EEF43592.1

Ricinus communis

EEF50924.1

Ricinus communis

EEF33440.1

Ricinus communis

Expression of Toxic Proteins or Polypeptides

The present disclosure describes a toxic protein, peptide, or small molecule that can be encoded by a recombinant microorganism. In some embodiments, the toxic protein, peptide, or small molecule is biosynthetically produced along with an insect pheromone.

In some embodiments, the recombinant microorganism expresses one or more nucleic acid molecules encoding a protein or polypeptide which is toxic to an insect. In some embodiments, the toxic protein or polypeptide is from an entomopathogenic organism. In some embodiments, the entomopathogenic organism is selected from Bacillus thuringiensis, Pseudomonas aenruginosa, and Serratia marcescens. In a particular embodiment, the nucleic acid molecule encodes a Bacillus thuringiensis toxin.

In some embodiments, a recombinant microorganism is engineered to express a metabolic pathway which, when expressed, produces a small molecule that is toxic to an insect.

In exemplary embodiments, an insect pheromone produced by a recombinant microorganism described herein may be used to attract a pest insect, and subsequently, the pest insect is eradicated with a toxic substance, such as a toxic protein, peptide, or small molecule, which has been co-produced by a recombinant microorganism described herein.

Biosynthesis of Pheromones Using a Recombinant Microorganism

As discussed above, in a first aspect, the present disclosure relates to a recombinant microorganism capable of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acyl-CoA. An illustrative embodiment of the first aspect is shown in FIG. 1. The blue lines designate biochemical pathways used to produce a saturated acyl-CoA, which acts as a substrate for unsaturated fatty-acyl CoA conversion. The substrate to unsaturated fatty acyl-CoA conversion can be performed by endogenous or exogenous enzymes in a host. Green lines indicate conversions catalyzed by an exogenous nucleic acid molecule encoding for an enzyme. Accordingly, in some embodiments, the conversion of a saturated fatty acyl-CoA to a mono- or poly-unsaturated fatty acyl-CoA is catalyzed by at least one desaturase, which is encoded by an exogenous nucleic acid molecule. In further embodiments, the conversion of the mono- or poly-unsaturated fatty acyl-CoA to a mono- or poly-unsaturated fatty alcohol is catalyzed by at least one reductase, which is encoded by an exogenous nucleic acid molecule. The dashed grey lines indicate downstream steps for the synthesis of pheromones, fragrances, flavors, and polymer intermediates, such as using an alcohol oxidase or oxidant to produce a mono- or poly-unsaturated fatty aldehyde, and an acetyl transferase or a chemical such as acetylchloride to produce a mono- or poly-unsaturated fatty acetate. The red crosses indicate deleted or down regulated pathways native to the host, which increase flux towards the engineered pathway.

Accordingly, in one embodiment, the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some embodiments, the saturated C₆-C₂₄fatty acyl-CoA can be produced using endogenous enzymes in the host microorganism. In other embodiments, the saturated C₆-C₂₄fatty acyl-CoA can be produced using one or more exogenous enzymes in the host microorganism.

As described above, a fatty acyl desaturase catalyzes the desaturation of the hydrocarbon chain on, e.g., a saturated fatty acyl-CoA molecule to generate a corresponding unsaturated fatty acyl CoA molecule. In some embodiments, an exogenous fatty acyl desaturase can be selected and expressed in a recombinant microorganism to catalyze the formation of at least one double bond in fatty acyl-CoA molecule having from 6 to 24 carbons in the hydrocarbon chain. Accordingly, in some embodiments, the fatty-acyl desaturase is a desaturase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

An exogenous fatty acyl desaturase described herein can be selected to catalyze the desaturation at a desired position on the hydrocarbon chain. Accordingly, in some embodiments, the fatty-acyl desaturase is capable of generating a double bond at position C5, C6, C7, C8, C9, C10, C11, C12, or C13, in the fatty acid or its derivatives, such as, for example, fatty acid CoA esters.

One or more than one fatty acyl-CoA desaturase can be expressed in the host to catalyze desaturation at multiple positions on the hydrocarbon chain. In some embodiments, the fatty acyl-CoA desaturase is heterologous to the host microorganism. Accordingly, various embodiments provide for recombinant microorganism comprised of at least one exogenous nucleic acid molecule, which encodes a fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA.

In one exemplary embodiment, the fatty-acyl desaturase is a Z11 desaturase. The Z11 fatty-acyl desaturase catalyze double bond formation between the 11^thand 12^thcarbons in the substrate relative to the carbonyl group. In various embodiments described herein, the Z11 desaturase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana. Further Z11-desaturases, or the nucleic acid sequences encoding them, can be isolated from Bombjx mori, Manduca sexta, Diatraea grandiosella, Earias insulana, Earias vittella, Plutella xylostella, Bombyx mori or Diaphania nitidalis. In exemplary embodiments, the Z11 desaturase comprises a sequence selected from GenBank Accession Nos. JX679209, JX964774, AF416738, AF545481, EU152335, AAD03775, AAF81787, and AY493438. In some embodiments, a nucleic acid sequence encoding a Z11 desaturase from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized. In some embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 9, 18, 24 and 26 from Trichoplusia ni. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 49 from Trichoplusia ni. In other embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 10 and 16 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 53 from Agrotis segetum. In some embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 11 and 23 from Thalassiosira pseudonana. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 50 and 51 from Thalassiosira pseudonana. In certain embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 12, 17 and 30 from Amyelois transitella. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 52 from Amyelois transitella. In further embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 13, 19, 25, 27 and 31 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 54 from Helicoverpa zea. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 39 from S. inferens. In some embodiments, the Z11 desaturase comprises an amino acid sequence set forth in GenBank Accession nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the Z11 desaturase comprises a chimeric polypeptide. In some embodiments, a complete or partial Z11 desaturase is fused to another polypeptide. In certain embodiments, the N-terminal native leader sequence of a Z11 desaturase is replaced by an oleosin leader sequence from another species. In certain embodiments, the Z11 desaturase comprises a sequence selected from SEQ ID NOs: 15, 28 and 29. In some embodiments, the Z11 desaturase comprises an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 78, 79 and 80.

In certain embodiments, the Z11 desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated product selected from Z11-13:Acyl-CoA, E11-13:Acyl-CoA, (Z,Z)-7,11-13:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, (E,E)-9,11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9.11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,Z)-9,11-15:Acyl-CoA, (Z,Z)-9,11-15:Acyl-CoA, Z11-16:Acyl-CoA, E11-16:Acyl-CoA, (E,Z)-6,11-16:Acyl-CoA, (E,Z)-7,11-16:Acyl-CoA, (E,Z)-8,11-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9,11-16:Acyl-CoA, (E,E)-11,13-16:Acyl-CoA, (E,Z)-11,13-16:Acyl-CoA, (Z,E)-11,13-16:Acyl-CoA, (Z,Z)-11,13-16:Acyl-CoA, (Z,E)-11,14-16:Acyl-CoA, (E,E,Z)-4,6,11-16:Acyl-CoA, (Z,Z,E)-7,11,13-16:Acyl-CoA, (E,E,Z,Z)-4,6,11,13-16:Acyl-CoA, Z11-17:Acyl-CoA, (Z,Z)-8,11-17:Acyl-CoA, Z11-18:Acyl-CoA, E11-18:Acyl-CoA, (Z,Z)-11,13-18:Acyl-CoA, (E,E)-11,14-18:Acyl-CoA, or combinations thereof.

In another exemplary embodiment, the fatty-acyl desaturase is a Z9 desaturase. The Z9 fatty-acyl desaturase catalyze double bond formation between the 9^thand 10^thcarbons in the substrate relative to the carbonyl group. In various embodiments described herein, the Z9 desaturase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Ostrinia furnacalis, Ostrinia nobilalis, Choristoneura rosaceana, Lampronia capitella, Helicoverpa assulta, or Helicoverpa zea. In exemplary embodiments, the Z9 desaturase comprises a sequence selected from GenBank Accession Nos. AY057862, AF243047, AF518017, EU152332, AF482906, and AAF81788. In some embodiments, a nucleic acid sequence encoding a Z9 desaturase is codon optimized. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 20 from Ostrinia furnacalis. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 58 from Ostrinia furnacalis. In other embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 21 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 59 from Lampronia capitella. In some embodiments, the Z9 desaturase comprises a nucleotide sequence set forth in SEQ ID NO: 22 from Helicoverpa zea. In some embodiments, the Z9 desaturase comprises an amino acid sequence set forth in SEQ ID NO: 60 from Helicoverpa zea.

In certain embodiments, the Z9 desaturase catalyzes the conversion of a fatty acyl-CoA into a monounsaturated or polyunsaturated product selected from Z9-11:Acyl-CoA, Z9-12:Acyl-CoA, E9-12:Acyl-CoA, (E,E)-7,9-12:Acyl-CoA, (E,Z)-7,9-12:Acyl-CoA, (Z,E)-7,9-12:Acyl-CoA, (Z,Z)-7,9-12:Acyl-CoA, Z9-13:Acyl-CoA, E9-13:Acyl-CoA, (E,Z)-5,9-13:Acyl-CoA, (Z,E)-5,9-13:Acyl-CoA, (Z,Z)-5,9-13:Acyl-CoA, Z9-14:Acyl-CoA, E9-14:Acyl-CoA, (E,Z)-4,9-14:Acyl-CoA, (E,E)-9.11-14:Acyl-CoA, (E,Z)-9,11-14:Acyl-CoA, (Z,E)-9,11-14:Acyl-CoA, (Z,Z)-9,11-14:Acyl-CoA, (E,E)-9,12-14:Acyl-CoA, (Z,E)-9,12-14:Acyl-CoA, (Z,Z)-9,12-14:Acyl-CoA, Z9-15:Acyl-CoA, E9-15:Acyl-CoA, (Z,Z)-6,9-15:Acyl-CoA, Z9-16:Acyl-CoA. E9-16:Acyl-CoA, (E,E)-9,11-16:Acyl-CoA, (E,Z)-9,11-16:Acyl-CoA, (Z,E)-9,11-16:Acyl-CoA, (Z,Z)-9.11-16:Acyl-CoA, Z9-17:Acyl-CoA, E9-18:Acyl-CoA, Z9-18:Acyl-CoA, (E,E)-5,9-18:Acyl-CoA, (E,E)-9,12-18:Acyl-CoA, (Z,Z)-9,12-18:Acyl-CoA, (Z,Z,Z)-3,6,9-18:Acyl-CoA, (E,E,E)-9,12,15-18:Acyl-CoA, (Z,Z,Z)-9,12,15-18:Acyl-CoA, or combinations thereof.

Desaturation of a saturated C₆-C₂₄fatty acyl-CoA can proceed through a plurality of reactions to produce a poly-unsaturated C₆-C₂₄fatty acyl-CoA. In some embodiments, the recombinant microorganism may express a bifunctional desaturase capable of catalyzing the formation at least two double bonds. In some embodiments, the recombinant microorganism may express more than one exogenous nucleic acid molecule encoding more than one fatty-acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding poly-unsaturated C₆-C₂₄fatty acyl-CoA. For example, the recombinant microorganism may express an exogenous nucleic acid molecule encoding a Z11 desaturase and another exogenous nucleic acid molecule encoding a Z9 desaturase. Thus, the resultant poly-unsaturated fatty acyl-CoA would have a double bond between the 9^thand 10^thcarbon and another double bond between the 11^thand 12^thcarbon.

In one embodiment, the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C₆-C₂₄fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl conjugase that catalyzes the conversion of a saturated or monounsaturated C₆-C₂₄fatty acyl-CoA to a corresponding polyunsaturated C₆-C₂₄fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding polyunsaturated C₆-C₂₄fatty alcohol.

In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a polyunsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated or monounsaturated C₆-C₂₄fatty acyl-CoA, wherein the recombinant microorganism expresses: (a) at least one exogenous nucleic acid molecule encoding a fatty-acyl desaturase and at least one exogenous nucleic acid molecule encoding a fatty acyl conjugase that catalyze the conversion of a saturated or monounsaturated C₆-C₂₄fatty acyl-CoA to a corresponding polyunsaturated C₆-C₂₄fatty acyl-CoA; and (b) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the polyunsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding polyunsaturated C₆-C₂₄fatty alcohol.

In another embodiment, the recombinant microorganism expresses at least two exogenous nucleic acid molecules encoding fatty-acyl desaturases and at least two exogenous nucleic acid molecules encoding fatty-acyl conjugases that catalyze the conversion of a saturated or monounsaturated C₄-C₂₄fatty acyl-CoA to a corresponding polyunsaturated C₆-C₂₄fatty acyl-CoA.

In certain embodiments, the conjugase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Cydia pomonella, Cydia nigricana, Lobesia botrana, Myelois cribrella, Plodia interpunctella, Dendrolimus punctatus, Lampronia capitella, Spodoptera litura, Amyelois transitella, Manduca sexta, Bombyx mori, Calendula officinalis, Trichosanthes kirilowii, Punica granatum, Momordica charantia, Impatiens balsamina, and Epiphyas postvittana. In exemplary embodiments, the conjugase comprises a sequence selected from GenBank Accession No. or Uniprot database: A0A059TBF5, A0A0M3L9E8, A0A0M3L9S4, A0A0M3LAH8, A0A0M3LAS8, A0A0M3LAH8, B6CBS4, XP_013183656.1, XP_004923568.2, ALA65425.1, NP_001296494.1, NP_001274330.1, Q4A181, Q75PL7. Q9FPP8, AY178444, AY178446, AF182521, AF182520, Q95UJ3.

As described above, a fatty acyl reductase catalyzes the reduction of a carbonyl group, e.g., on an unsaturated fatty acyl-CoA molecule to generate a corresponding unsaturated fatty acid molecule. In some embodiments, the fatty alcohol forming fatty acyl CoA reductase is heterologous to the microorganism. Accordingly, various embodiments provide for recombinant microorganism comprised of at least one exogenous nucleic acid molecule, which encodes a fatty alcohol forming fatty acyl reductase that catalyzes the reduction of a carbonyl group on an unsaturated fatty acyl-CoA molecule to generate a corresponding unsaturated fatty acid molecule.

In some embodiments, the fatty acyl reductase is from an organism of the species Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera. In some embodiments, a nucleic acid sequence encoding a fatty-acyl reductase is codon optimized. In some embodiments, the fatty acyl reductase comprises a sequence set forth in SEQ ID NO: 1 from Agrotis segetum. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises a sequence set forth in SEQ ID NO: 2 from Spodoptera littoralis. In other embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises a sequence selected from SEQ ID NOs: SEQ ID NOs: 3, 32, 40, 72, 74, 76 and 81. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 55 from Agrotis segetum. In other embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 56 from Spodoptera littoralis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 41 and 57 from Helicoverpa armigera. In some embodiments, the fatty acyl reductase comprises an amino acid sequence selected from SEQ ID NOs: 73 and 82 from Spodoptera exigua. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 75 from Euglena gracilis. In some embodiments, the fatty acyl reductase comprises an amino acid sequence set forth in SEQ ID NO: 77 from Yponomeuta evonymellus.

In some embodiments, the production of unsaturated fatty alcohols in a recombinant microorganism comprises the expression of one or more mutant FARs. In certain embodiments, Helicoverpa amigera fatty acyl-CoA reductase (HaFAR) variants are provided which show a net increase in fatty alcohol produced compared to the wild type Helicoverpa amigera fatty acyl-CoA reductase encoded by an amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the increased enzymatic activity is a net activity increase in amount of fatty alcohol produced relative to the amount of fatty alcohol produced by a wild type enzymatic activity of HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, a wild type HaFAR comprises a nucleotide sequence set forth in SEQ ID NO: 90. In some embodiments, a variant of a wild type HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41 comprises point mutations at the following positions: S60X, S195X, S298X. S378X. S394X, S418X, and S453X, wherein X comprises the amino acids F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W, R. In some embodiments, a variant of a wild type HaFAR encoded by an amino acid sequence set forth in SEQ ID NO: 41 comprises a combination of point mutations selected from mutations at the following amino acid positions: S60X, S195X, S298X, S378X, S394X, S418X, and S453X, wherein X comprises the amino acids F, L, M, I, V, P, T, A, Y, K, H, N, Q, K, D, E, C, W, R. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises an amino acid sequence selected from SEQ ID NOs: 42-48. In some embodiments, the fatty acyl reductase is a mutated fatty acyl reductase and comprises nucleotide sequence selected from SEQ ID NOs: 83-89.

In exemplary embodiments, the fatty-acyl reductase catalyzes the conversion of a mono- or poly-unsaturated fatty acyl-CoA into a fatty alcohol product selected from (Z)-3-hexenol, (Z)-3-nonenol, (Z)-5-decenol, (E)-5-decenol. (Z)-7-dodecenol, (E)-8-dodecenol, (Z)-8-dodecenol, (Z)-9-dodecenol, (Z)-9-tetradecenol, (Z)-9-hexadcenol, (Z)-11-tetradecenol, (Z)-7-hexadecenol, (Z)-11-hexadecenol, (E)-11-tetradecenol, or (Z,Z)-11,13-hexadecadienol, (11Z,13E)-hexadecadienol, (E,E)-8,10-dodecadienol, (E,Z)-7,9-dodecadienol, (Z)-13-octadecenol, or combinations thereof.

In some embodiments, a recombinant microorganism described herein can include a plurality of fatty acyl reductases. Accordingly, in such embodiments, the recombinant microorganism expresses at least two exogenous nucleic acid molecules, which encode fatty-acyl reductases that catalyze the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.

In a further embodiment, the disclosure provides a recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the recombinant microorganism comprises: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C₄-C₂₂fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA substrate in that cycle, and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C18 fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C18 fatty alcohol. In some embodiments, the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase. In some embodiments, the fatty acyl desaturase has 95% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879. ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In some embodiments, the acyl-CoA oxidase is selected from Table 5a. In other embodiments, the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase. In further embodiments, the fatty alcohol forming fatty acyl reductase has 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82. In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.

In some embodiments, the coexpression of a wax esterase would allow the storage of fatty alcohols and fatty acids in a 1:1 ratio. In combination with TAG storage that could lead to interesting ratios of TAG and fatty alcohols which could subsequently be used for different product streams. Examples for waxester synthases: Homo sapiens AWAT2 (XM_011530876.2), Mus musculus (AAT68766.1) Euglena gracilis WS (ADI60058.1), Euglena gracilis WSD2 (BAV82975.1), Euglena gracilis WSD5 (BAV82978.1).

In some preferred embodiments, one or more genes of the microbial host encoding acyl-CoA oxidases are deleted or down-regulated to eliminate or reduce the truncation of desired fatty acyl-CoAs beyond a desired chain-length. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y. lipolytica POX1 (YALI0E32835g), Y. lipolytica POX2 (YALI0F10857g), Y. lipolytica POX3 (YALI0D24750g), Y. lipolytica POX4 (YALI0E27654g), Y. lipolytica POX5 (YALI0C23859g), Y. lipolytica POX6 (YALI0E06567g); S. cerevisiae POX1 (YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146. CTRG_02721, M12161).

In some embodiments, a recombinant microorganism capable of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol, fatty aldehyde and/or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid is provided, wherein the recombinant microorganism expresses one or more acyl-CoA oxidase enzymes, and wherein the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous acyl-CoA oxidase enzymes. In some embodiments, the one or more acyl-CoA oxidase enzymes being expressed are different from the one or more endogenous acyl-CoA oxidase enzymes being deleted or downregulated. In other embodiments, the one or more acyl-CoA oxidase enzymes that are expressed regulate chain length of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol, fatty aldehyde and/or fatty acetate. In other embodiments, the one or more acyl-CoA oxidase enzymes being expressed are selected from Table 5a.

In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g. S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645. Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.

In some preferred embodiments, one or more genes of the microbial host encoding acylglycerol lipases (mono-, di-, or triacylglycerol lipases) and sterol ester esterases are deleted or downregulated and replaced with one or more acylglycerol lipases which prefer long chain acylglycerol substrates. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598. Candida CTRG_01138. Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426. Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049. Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.

In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6). Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK5), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK12).

In some embodiments, the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA, E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z15-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA, E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E14-16:Acyl-CoA, Z13E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z1Z13-16:Acyl-CoA, and Z13Z15-18:Acyl-CoA. In further embodiments, the mono- or poly-unsaturated ≤C₁₈fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH, and Z13-18:OH.

In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈8 fatty alcohol into a corresponding ≤C₁₈fatty aldehyde. In some preferred embodiments, the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C₄HN78, A. calcoaceticus N9DA85. A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A1117N158, and Nostoc punctiforme YP_001865324. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde. In some preferred embodiments, the ≤C₁₈fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.

In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty acetate. In some embodiments, the acetyl transferase is selected from Table 5d. In some preferred embodiments, the ≤C18 fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, and Z11-16:Ac.

In some embodiments, the recombinant microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase, and an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty acetate. In some preferred embodiments, the mono- or poly-unsaturated ≤C18 fatty aldehyde and ≤C₁₈fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.

In a further embodiment, the disclosure provides a recombinant Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the recombinant Yarrowia lipolytica microorganism comprises: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.

In some embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3). YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK1) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2). In other preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1). YALI0F10857g (POX2). YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH). YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3). YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B3838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8).YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK1) and YALI0A20130g (Alk12), and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).

In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate. In some embodiments, the acetyl transferase is selected from Table 5d. In some embodiments, the C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, and Z13-18:Ac.

In some embodiments, the recombinant Yarrowia lipolytica microorganism further comprises: at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde and C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac. E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z1Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

In some embodiments, the disclosure provides a method of engineering a Yarrowia lipolytica microorganism that is capable of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the method comprises introducing into the Yarrowia lipolytica microorganism the following: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some embodiments, the microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.

In some embodiments, the disclosure provides a method of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol, fatty aldehyde or fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating a recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol, fatty aldehyde or fatty acetate. In some embodiments, the method further comprises a step of recovering the mono- or poly-unsaturated C₆-C₂₄fatty alcohol, fatty aldehyde or fatty acetate. In further embodiments, the recovery step comprises distillation. In yet further embodiments, the recovery step comprises membrane-based separation.

As discussed above, in a second aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of C₆-C₂₄fatty acid. An illustrative embodiment of the second aspect is shown in FIG. 2. The blue lines designate biochemical pathways endogenous to the host, e.g., pathways for converting an n-alkane, fatty alcohol, or fatty aldehyde to a fatty acid, or the conversion of a fatty acid to fatty-acyl-CoA, acetyl-CoA, or dicarboxylic acid. The substrate to unsaturated fatty acid conversion can be performed by endogenous or exogenous enzymes in a host. Yellow lines indicate conversions catalyzed by an exogenous nucleic acid molecule encoding for an enzyme. Accordingly, in some embodiments, the conversion of a saturated fatty acid to a saturated fatty acyl-ACP can be catalyzed by at least one saturated fatty acyl-ACP synthetase, wherein the fatty acyl-ACP synthetase is encoded by an exogenous nucleic acid molecule. In further embodiments, the conversion of the saturated fatty acyl-ACP to a mono- or poly-unsaturated fatty acyl-ACP can be catalyzed by at least one fatty acyl-ACP desaturase, wherein the fatty acyl-ACP desaturase is encoded by an exogenous nucleic acid molecule. In still further embodiments, the mono- or poly-unsaturated fatty acyl-ACP can be elongated by at least 2 carbons relative using a fatty acid synthase complex and a carbon source, e.g., malonyl-ACP. In one such embodiment, the conversion of the mono- or poly-unsaturated fatty acyl-ACP to a corresponding two carbon elongated mono- or poly-unsaturated fatty acyl-ACP can be catalyzed by at least one fatty acid synthase complex, wherein the fatty acid synthase complex is encoded by one or more exogenous nucleic acid molecules. In yet further embodiments, the conversion of the elongated mono- or poly-unsaturated fatty acyl-ACP to a mono- or poly-unsaturated fatty aldehyde can be catalyzed by a fatty aldehyde forming fatty acyl reductase, wherein the fatty aldehyde forming fatty acyl reductase is encoded by an exogenous nucleic acid molecule. In some embodiments, the mono- or poly-unsaturated fatty aldehyde can be converted to a corresponding mono- or poly-unsaturated fatty alcohol, wherein the substrate to product conversion is catalyzed by a dehydrogenase, wherein the dehydrogenase is encoded by an endogenous or exogenous nucleic acid molecule. The dashed lines indicate downstream steps of the disclosure, such as utilizing an acetyl transferase or metathesis, or subsequent chemical transformations to produce functionalized pheromones. The red crosses indicate deleted or down regulated pathways native to the host, which increase flux towards the engineered pathway.

In one embodiment, the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding an acyl-ACP synthetase that catalyzes the conversion of a C₆-C₂₄fatty acid to a corresponding saturated C₆-C₂₄fatty acyl-ACP; (b) at least one exogenous nucleic acid molecule encoding a fatty-acyl-ACP desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-ACP to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP; (c) one or more endogenous or exogenous nucleic acid molecules encoding a fatty acid synthase complex that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP from (b) to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP with a two carbon elongation relative to the product of (b); (d): at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-ACP from (c) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty aldehyde; and (e) at least one endogenous or exogenous nucleic acid molecule encoding a dehydrogenase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde C₆-C₂₄from (d) into a corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some embodiments, the C₆-C₂₄fatty acid can be produced using endogenous enzymes in the host microorganism. In other embodiments, the saturated C₆-C₂₄fatty acid can be produced by one or more exogenous enzymes in the host microorganism.

In some embodiments, the recombinant microorganism disclosed herein includes an acyl-ACP synthetase to catalyze the conversion of a C₆-C₂₄fatty acid to a corresponding saturated C₆-C₂₄fatty acyl-ACP. In some embodiments the acyl-ACP synthetase is a synthetase capable of utilizing a fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. In exemplary embodiments, the recombinant microorganism can include a heterologous the acyl-ACP synthetase from an organism of the species Vibrio harveyi, Rhodotorula glutinis, or Yarrowia lipolytica.

In some embodiments, the recombinant microorganism includes a fatty acyl-ACP desaturase. In some embodiments, the fatty acyl-ACP desaturase is a soluble desaturase. In other embodiments, the fatty-acyl-ACP desaturase is from an organism of the species Pelargonium hortorum, Asclepias syriaca, or Uncaria tomentosa.

In some embodiments, the recombinant microorganism includes a fatty acid synthase complex. In some embodiments, the one or more nucleic acid molecules encoding the fatty acid synthase complex are endogenous nucleic acid molecules. In other embodiments, the one or more nucleic acid molecules encoding a fatty acid synthase complex are exogenous nucleic acid molecules.

In some embodiments, the recombinant microorganism disclosed herein includes a fatty aldehyde forming fatty-acyl reductase which catalyzes the conversion of a C₆-C₂₄fatty acyl-ACP to the corresponding C₆-C₂₄fatty aldehyde. In exemplary embodiments, the fatty aldehyde forming fatty-acyl reductase is from an organism of the species Pelargonium hortorum, Asclepias syriaca, and Uncaria tomentosa. In some embodiments, the recombinant microorganism includes a dehydrogenase to convert the unsaturated fatty aldehyde to a corresponding unsaturated fatty alcohol. In some embodiments, the nucleic acid molecule encoding the dehydrogenase is endogenous to the recombinant microorganism. In other embodiments, the nucleic acid molecule encoding a dehydrogenase is exogenous to the recombinant microorganism. In exemplary embodiments, the endogenous or exogenous nucleic acid molecule encoding a dehydrogenase is isolated from organisms of the species Saccharomyces cerevisiae, Escherichia coli, Yarrowia lipolytica, or Candida tropicalis.

As discussed above, in a third aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of C₆-C₂₄fatty acid. An illustrative embodiment of the second aspect is shown in FIG. 3. The blue lines designate biochemical pathways endogenous to the host, e.g., pathways for converting an n-alkane, fatty alcohol, or fatty aldehyde to a fatty acid, or the conversion of a fatty acid to fatty-acyl-CoA, acetyl-CoA, or dicarboxylic acid. The substrate to unsaturated fatty acid conversion can be performed by endogenous or exogenous enzymes in a host. Yellow lines indicate conversions catalyzed by an exogenous nucleic acid molecule encoding for an enzyme. Accordingly, in some embodiments, the conversion of a saturated fatty acid to a saturated fatty acyl-ACP can be catalyzed by at least one saturated fatty acyl-ACP synthetase, wherein the fatty acyl-ACP synthetase is encoded by an exogenous nucleic acid molecule. The non-native saturated fatty acyl-ACP thioesters create a substrate suitable for desaturation and distinct from CoA-thioesters used for beta-oxidation or fatty acid elongation. In further embodiments, the conversion of the saturated fatty acyl-ACP to a mono- or poly-unsaturated fatty acyl-ACP can be catalyzed by at least one fatty acyl-ACP desaturase, wherein the fatty acyl-ACP desaturase is encoded by an exogenous nucleic acid molecule. In still further embodiments, the mono- or poly-unsaturated fatty acyl-ACP can be converted to a corresponding mono- or poly-unsaturated fatty acid by a fatty-acyl-ACP thioesterase. In a particular embodiment, soluble fatty acyl-ACP thioesterases can be used to release free fatty acids for reactivation to a CoA thioester. Fatty acyl-ACP thioesterases including Q41635, Q39473, P05521.2, AEM72519. AEM72520, AEM72521, AEM72523, AAC49784, CAB60830, EER87824, EER96252, ABN54268, AAO77182, CAH09236, ACL08376, and homologs thereof may be used. In an additional embodiment, the mono- or poly-unsaturated fatty acyl-CoA can be elongated by at least 2 carbons relative using an elongase and a carbon source, e.g., malonyl-ACP. In yet further embodiments, the conversion of the elongated mono- or poly-unsaturated fatty acyl-CoA to a mono- or poly-unsaturated fatty alcohol can be catalyzed by a fatty alcohol forming fatty acyl reductase, wherein the fatty alcohol forming fatty acyl reductase is encoded by an exogenous nucleic acid molecule. The dashed lines indicate downstream steps of the disclosure, such as utilizing an acetyl transferase or metathesis, or subsequent chemical transformations to produce functionalized pheromones. The red crosses indicate deleted or down regulated pathways native to the host, which increase flux towards the engineered pathway.

The fatty alcohols produced as taught herein can be further converted to produce downstream products such as insect pheromones, fragrances, flavors, and polymer intermediates, which utilize aldehydes or acetate functional groups. Thus, in some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of a C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde. In other embodiments, the recombinant microorganism can further comprise at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of a C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate. In certain embodiments, the acetyl transferase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Candida glabrata, Saccharomyces cerevisiae, Danaus plexippus, Heliotis virescens, Bombyx mori, Agrotis ipsilon, Agrotis segetum, Euonymus alatus. Homo sapiens, Lachancea thermotolerans and Yarrowia lipolytica. In exemplary embodiments, the acetyl transferase comprises a sequence selected from GenBank Accession Nos. AY242066, AY242065, AY242064, AY242063, AY242062, EHJ65205, ACX53812, NP_001182381, EHJ65977, EHJ68573, KJ579226, GU594061 KTA99184.1, AIN34693.1, AY605053, XP_002552712.1, XP_503024.1, and XP_505595.1.

Recombinant Microorganism

The disclosure provides microorganisms that can be engineered to express various exogenous enzymes.

In various embodiments described herein, the recombinant microorganism is a eukaryotic microorganism. In some embodiments, the eukaryotic microorganism is a yeast. In exemplary embodiments, the yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma.

The present inventors have discovered that oleaginous yeast, such as Candida and Yarrowia, have a surprisingly high tolerance to the C₆-C₂₄fatty alcohol substrates and products. Accordingly, in one such exemplary embodiment, the recombinant microorganism of the invention is an oleaginous yeast. In further embodiments, the oleaginous yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium. Cryptococcus, Trichosporon, and Lipomyces. In even further embodiments, the oleaginous yeast is a member of a species selected from Yarrowia lipolytica, Candida tropicalis, Rhodosporidium toruloides, Lipomyces starkey, L. lipoferus, C. revkaufi. C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T. culaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.

In some embodiments, the recombinant microorganism is a prokaryotic microorganism. In exemplary embodiments, the prokaryotic microorganism is a member of a genus selected from the group consisting of Escherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium.

In some embodiments, the recombinant microorganism is used to produce a mono- or poly-unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate disclosed herein.

Accordingly, in another aspect, the present inventions provide a method of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate using a recombinant microorganism described herein. In one embodiment, the method comprises cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until the mono- or poly-unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate is produced. In some embodiments, the method comprises cultivating the recombinant microorganism described herein in a culture medium containing a feedstock that provides a carbon source adequate for the production of a mono- or poly-unsaturated ≤C₁₈fatty alcohol, fatty aldehyde or fatty acetate. In a further embodiment, the mono- or poly-unsaturated ≤C₁₈fatty alcohol, aldehyde, or acetate is recovered. Recovery can be by methods known in the art, such as distillation, membrane-based separation gas stripping, solvent extraction, and expanded bed adsorption.

In some embodiments, the feedstock comprises a carbon source. In various embodiments described herein, the carbon source may be selected from sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocellulose, proteins, carbon dioxide, and carbon monoxide. In a further embodiment, the sugar is selected from the group consisting of glucose, fructose, and sucrose.

Methods of engineering microorganisms that are capable of producing mono- or poly-unsaturated C₆-C₂₄fatty alcohols, fatty aldehydes and/or fatty acetates

In one aspect, the present disclosure provides a method of engineering a microorganism that is capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the method comprises introducing into a microorganism the following: (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C₄-C₂₂fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA substrate in that cycle; and (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C₁₈fatty alcohol. In some embodiments, the microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.

In some embodiments, the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase. In some preferred embodiments, the fatty acyl desaturase has 95% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1 and NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1. In further embodiments, the acyl-CoA oxidase is selected from Table 5a. In yet further embodiments, the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua. Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase. In further embodiments, the fatty alcohol forming fatty acyl reductase has 90% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82. In some embodiments, the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica. Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.

In some embodiments, the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA, E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z2-16:Acyl-CoA, Z4-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z15-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA. E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E14-16:Acyl-CoA, Z13E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z11Z13-16:Acyl-CoA, and Z13Z15-18:Acyl-CoA. In further embodiments, the mono- or poly-unsaturated ≤C₁₈fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH. Z9-16:OH, and Z13-18:OH.

In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C₁₈fatty acyl-CoA. In some embodiments, the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT). In some preferred embodiments, the acyltransferase is selected from Table 5b.

In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates. In some embodiments, the acylglycerol lipase is selected from Table 5c.

In some embodiments, the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ≤C₁₈fatty alcohol. In further embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from: (i) one or more acyl-CoA oxidase; (ii) one or more acyltransferase; (iii) one or more acylglycerol lipase and/or sterol ester esterase; (iv) one or more (fatty) alcohol dehydrogenase; (v) one or more (fatty) alcohol oxidase; and (vi) one or more cytochrome P450 monooxygenase.

In some embodiments, the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.

In some embodiments, the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C4520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR08c, S. cerevisiae YKL094W. S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398, Candida CTRG_00057, Candid CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443. Candida CTRG_01683, and Candida CTRG_04630.

In some embodiments, the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6), Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and K. lipolytica YALI0A20130g (ALK12).

In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde. In some preferred embodiments, the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A117N158, and Nostoc punctiforme YP_001865324. In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde. In some preferred embodiments, the ≤C₁₈fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.

In some embodiments, the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty acetate. In some embodiments, the acetyl transferase is selected from Table 5d. In some preferred embodiments, the ≤C₁₈fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, and Z11-16:Ac.

In some embodiments, the method further comprises introducing into the microorganism: at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase, and an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty acetate. In some preferred embodiments, the mono- or poly-unsaturated ≤C₁₈fatty aldehyde and ≤C₁₈fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.

In some embodiments, the mono- or poly-unsaturated ≤C₁₈fatty alcohol is converted into a corresponding ≤C₁₈fatty aldehyde using chemical methods. In further embodiments, the chemical methods are selected from TEMPO-bleach. TEMPO-copper-air, TEMPO-PhI(OAc), Swern oxidation and noble metal-air. In some embodiments, the mono- or poly-unsaturated ≤C₁₈fatty alcohol is converted into a corresponding ≤C₁₈fatty acetate using chemical methods. In further embodiments, the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated ≤C₁₈fatty alcohol to the corresponding ≤C₁₈fatty acetate.

In another aspect, the present disclosure provides methods of engineering a Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the recombinant Yarrowia lipolytica microorganism comprises: (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; and (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.

In some embodiments, the method further comprises introducing into the Yarrowia lipolytica microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C₆-C₂₄fatty alcohol. In some preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F0857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2). In other preferred embodiments, the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzyme selected from the following: (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2). YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6). YALI0E07766g (ADH7); (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1); (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).

In some embodiments, the method further comprises introducing into the Yarrowia lipolytica microorganism at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde. In some embodiments, the alcohol dehydrogenase is selected from Table 3a. In some embodiments, the C₆-C₂₄fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

In some embodiments, the method further comprises introducing into the Yarrowia lipolytica microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate. In some embodiments, the acetyl transferase is selected from Table 5d. In some embodiments, the C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z3-16:Ac, and Z13-18:Ac.

In some embodiments, the method further comprises introducing into the Yarrowia lipolytica microorganism: at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde; and at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde and C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.

In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is converted into a corresponding C₆-C₂₄fatty aldehyde using chemical methods. In further embodiments, the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air. TEMPO-PhI(OAc)₂. Swern oxidation and noble metal-air. In some embodiments, the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is converted into a corresponding C₆-C₂₄fatty acetate using chemical methods. In further embodiments, the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N, N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated C₆-C₂₄fatty alcohol to the corresponding C₆-C₂₄fatty acetate.

Enzyme Engineering

The enzymes in the recombinant microorganism can be engineered to improve one or more aspects of the substrate to product conversion. Non-limiting examples of enzymes that can be further engineered for use in methods of the disclosure include a desaturase (e.g., a fatty acyl-CoA desaturase or fatty acyl-ACP desaturase), a fatty alcohol forming fatty acyl reductase, an acyl-ACP synthetase, a fatty acid synthetase, a fatty acid synthase complex, an acetyl transferase, dehydrogenase, and an alcohol oxidase, and combinations thereof. These enzymes can be engineered for improved catalytic activity, improved selectivity, improved stability, improved tolerance to various fermentations conditions (temperature, pH, etc.), or improved tolerance to various metabolic substrates, products, by-products, intermediates, etc.

Desaturase enzymes can be engineered for improved catalytic activity in the desaturation of an unsaturated substrate, for improved hydrocarbon selectivity, for improved selectivity of a Z product over an E product, or an E product over a Z product. For example, the Z9 fatty-acyl desaturase can be engineered to improve the yield in the substrate to product conversion of a saturated fatty acyl-CoA to the corresponding unsaturated fatty acyl-CoA, and, in addition or in the alternative, to improve selectivity of the desaturation at the 9 position to produce a corresponding Z-9 fatty acyl-CoA. In further non-limiting examples, the fatty acyl-ACP synthetase can be engineered for improved ACP ligation activity; a fatty acid synthase complex enzyme can be engineered for improved catalytic activity of elongation of a fatty acid substrate; a fatty alcohol forming fatty acyl-reductase can be engineered for improved catalytic activity in the reduction of a fatty acyl-CoA to a corresponding fatty alcohol: a fatty aldehyde forming fatty acyl-reductase can be engineered for improved catalytic activity in the reduction of a fatty acyl-ACP to a corresponding fatty aldehyde: a dehydrogenase can be engineered for improved catalytic activity in the conversion of a fatty acyl-ACP to a corresponding fatty alcohol; an alcohol oxidase can be engineered for improved catalytic activity in the conversion of a fatty alcohol into a corresponding fatty aldehyde; and an acetyl transferase can be engineered for improved catalytic activity in the conversion of a fatty alcohol into a corresponding fatty acetate.

The term “improved catalytic activity” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured relative to a comparable non-engineered enzyme, such as a non-engineered desaturase (e.g. fatty acyl-CoA desaturase or fatty acyl-ACP desaturase), fatty alcohol or aldehyde forming fatty-acyl reductase, acyl-ACP synthetase, fatty acid synthetase, fatty acid synthase complex, acyl transferase, dehydrogenase, or an alcohol oxidase enzyme. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Mutations can be introduced into a desaturase (e.g. fatty acyl-CoA desaturase or fatty acyl-ACP desaturase), a fatty alcohol or aldehyde forming fatty-acyl reductase, a acyl-ACP synthetase, a fatty acid synthetase, a fatty acid synthase complex, a acyl transferase, a dehydrogenase, or an alcohol oxidase enzyme resulting in engineered enzymes with improved catalytic activity. Methods to increase enzymatic activity are known to those skilled in the art. Such techniques can include increasing the expression of the enzyme by increasing plasmid copy number and/or use of a stronger promoter and/or use of activating riboswitches, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the K_Mfor the substrate, or by directed evolution. See. e.g., Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003).

Metabolic Engineering—Enzyme Overexpression and Gene Deletion/Downregulation for Increased Pathway Flux

In various embodiments described herein, the exogenous and endogenous enzymes in the recombinant microorganism participating in the biosynthesis pathways described herein may be overexpressed.

The terms “overexpressed” or “overexpression” refers to an elevated level (e.g., aberrant level) of mRNAs encoding for a protein(s), and/or to elevated levels of protein(s) in cells as compared to similar corresponding unmodified cells expressing basal levels of mRNAs or having basal levels of proteins. In particular embodiments, mRNA(s) or protein(s) may be overexpressed by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold or more in microorganisms engineered to exhibit increased gene mRNA, protein, and/or activity.

In some embodiments, a recombinant microorganism of the disclosure is generated from a host that contains the enzymatic capability to synthesize a substrate fatty acid. In this specific embodiment it can be useful to increase the synthesis or accumulation of a fatty acid to, for example, increase the amount of fatty acid available to an engineered fatty alcohol production pathway.

In some embodiments, it may be useful to increase the expression of endogenous or exogenous enzymes involved in the fatty alcohol, aldehyde, or acetate production pathway to increase flux from the fatty acid to the fatty alcohol, aldehyde, or acetate, thereby resulting in increased synthesis or accumulation of the fatty alcohol, aldehyde, or acetate.

In some embodiments, it may be useful to increase the expression of endogenous or exogenous enzymes to increase intracellular levels of a coenzyme. In one embodiment, the coenzyme is NADH. In another embodiment, the coenzyme is NADPH. In one embodiment, the expression of proteins in the pentose phosphate pathway is increased to increase the intracellular levels of NADPH. The pentose phosphate pathway is an important catabolic pathway for supplying reduction equivalents and an important anabolic pathway for biosynthesis reactions. In one embodiment, a glucose-6-phosphate dehydrogenase that converts glucose-6-phosphate to 6-phospho D-glucono-1,5-lactone is overexpressed. In some embodiments, the glucose-6-phosphate dehydrogenase is ZWF1 from yeast. In another embodiment, the glucose-6-phosphate dehydrogenase is ZWF1 (YNL241C) from Saccharomyces cerevisiae. In one embodiment, a glucose-6-phosphate-1-dehydrogenase that converts D-glucopyranose-6-phosphate to 6-phospho D-glucono-1,5-lactone is overexpressed. In another embodiment, the glucose-6-phosphate-1-dehydrogenase is zwf from bacteria. In certain embodiments, the glucose-6-phosphate-1-dehydrogenase is zwf (NP_416366) from E. coli. In one embodiment, a 6-phosphogluconolactonase that converts 6-phospho D-glucono-1,5-lactone to D-gluconate 6-phosphate is overexpressed. In some embodiments, the 6-phosphogluconolactonase is SOL3 of yeast. In certain embodiments, the 6-phosphogluconolactonase is SOL3 (NP_012033) of Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconolactonase is SOL4 of yeast. In certain embodiments, the 6-phosphogluconolactonase is SOL4 (NP_011764) of Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconolactonase is pgl of bacteria. In certain embodiments, the 6-phosphogluconolactonase is pgl (NP_415288) of E. coli. In one embodiment, a 6-phosphogluconate dehydrogenase that converts D-gluconate 6-phosphate to D-ribulose 5-phosphate is overexpressed. In some embodiments, the 6-phosphogluconate dehydrogenase is GND1 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND1 (YHR183W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is GND2 from yeast. In certain embodiments, the 6-phosphogluconate dehydrogenase is GND2 (YGR256W) from Saccharomyces cerevisiae. In some embodiments, the 6-phosphogluconate dehydrogenase is gnd from bacteria. In certain embodiments, the 6-phosphogluconate dehydrogenase is gnd (NP_416533) from E. coli. In one embodiment, a transaldolase that interconverts D-glyceraldehyde 3-phosphate and D-sedoheptulose 7-phosphate to β-D-fructofuranose 6-phosphate and D-erythrose 4-phosphate is overexpressed. In some embodiments, the transaldolase is TAL1 of yeast. In certain embodiments, the transaldolase is TAL1 (NP_013458) of Saccharomyces cerevisiae. In some embodiments, the transaldolase is NQM1 of yeast. In certain embodiments, the transaldolase is NQM1 (NP_011557) of Saccharomyces cerevisiae. In some embodiments, the transaldolase is tal of bacteria. In certain embodiments, the transaldolase is talB (NP_414549) of E. coli. In certain embodiments, the transaldolase is talA (NP_416959) of E. coli. In one embodiment, a transketolase that interconverts D-erythrose 4-phosphate and D-xylulose 5-phosphate to β-D-fructofuranose 6-phosphate and D-glyceraldehyde 3-phosphate and/or interconverts D-sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate to D-ribose 5-phosphate and D-xylulose 5-phosphate is overexpressed. In some embodiments, the transketolase is TKL1 of yeast. In certain embodiments, the transketolase is TKL1 (NP_015399) of Saccharomyces cerevisiae. In some embodiments, the transketolase is TKL2 of yeast. In some embodiments, the transketolase is TKL2 (NP_009675) of Saccharomyces cerevisiae. In some embodiments, the transketolase is tkt of bacteria. In certain embodiments, the transketolase is tktA (YP_026188) of E. coli. In certain embodiments, the transketolase is tktB (NP_416960) of E. coli. In one embodiment, a ribose-5-phosphate ketol-isomerase that interconverts D-ribose 5-phosphate and D-ribulose 5-phosphate is overexpressed. In some embodiments, the ribose-5-phosphate ketol-isomerase is RKI1 of yeast. In certain embodiments, the ribose-5-phosphate ketol-isomerase is RKI1 (NP_014738) of Saccharomyces cerevisiae. In some embodiments, the ribose-5-phosphate isomerase is rpi of bacteria. In certain embodiments, the ribose-5-phosphate isomerase is rpiA (NP_417389) of E. coli. In certain embodiments, the ribose-5-phosphate isomerase is rpiB (NP_418514) of E. coli. In one embodiment, a D-ribulose-5-phosphate 3-epimerase that interconverts D-ribulose 5-phosphate and D-xylulose 5-phosphate is overexpressed. In some embodiments, the D-ribulose-5-phosphate 3-epimerase is RPE1 of yeast. In certain embodiments, the D-ribulose-5-phosphate 3-epimerase is RPE1 (NP_012414) of Saccharomyces cerevisiae. In some embodiments, the D-ribulose-5-phosphate 3-epimerase is rpe of bacteria. In certain embodiments, the D-ribulose-5-phosphate 3-epimerase is rpe (NP_417845) of E. coli.

In one embodiment, the expression of an NADP+-dependent isocitrate dehydrogenase is increased to increase intracellular levels of a coenzyme. In one embodiment, an NADP+ dependent isocitrate dehydrogenase oxidizes D-threo-isocitrate to 2-oxoglutarate with concomitant generation of NADPH. In another embodiment, an NADP+ dependent isocitrate dehydrogenase oxidizes D-threo-isocitrate to 2-oxalosuccinate with concomitant generation of NADPH. In some embodiments, the NADP+-dependent isocitrate dehydrogenase is IDP from yeast. In certain embodiments, the NADP+-dependent isocitrate dehydrogenase is IDP2 (YLR174W) from Saccharomyces cerevisiae. In some embodiments, the NADP+-dependent isocitrate dehydrogenase is icd from bacteria. In certain embodiments, the NADP+-dependent isocitrate dehydrogenase is icd (NP_415654) from E. coli.

In some embodiments, the expression of a malic enzyme that decarboxylates malate to pyruvate with concomitant generation of NADH or NADPH is increased to increase intracellular levels of a coenzyme. In one embodiment, the malic enzyme is NAD+ dependent. In another embodiment, the malic enzyme is NADP+ dependent. In one embodiment, the malic enzyme is an NAD+ dependent malate dehydrogenase from bacteria. In some embodiments, the NAD+ dependent malate dehydrogenase is maeA (NP_415996) from E. coli. In some embodiments, the NAD+ dependent malate dehydrogenase is macE (CAQ68119) from Lactobacillus casei. In another embodiment, the malic enzyme is a mitochondrial NAD+ dependent malate dehydrogenase from yeast. In some embodiments, the NAD+ dependent malate dehydrogenase is MAE1 (YKL029C) from S. cerevisiae. In another embodiment, the malic enzyme is a mitochondrial NAD+ dependent malate dehydrogenase from a parasitic nematode. In some embodiments, the NAD+ dependent malate dehydrogenase is M81055 from Ascaris suum. In one embodiment, the malic enzyme is an NADP+ dependent malate dehydrogenase from bacteria. In some embodiments, the NADP+ dependent malate dehydrogenase is macB (NP_416958) from E. coli. In one embodiment, the malic enzyme is an NADP+ dependent malate dehydrogenase from corn. In some embodiments, the NADP+ dependent malate dehydrogenase is me 1 from Zea mays.

In some embodiments, the expression of an aldehyde dehydrogenase that oxidizes an aldehyde to a carboxylic acid with concomitant generation of NADH or NADPH is increased to increase intracellular levels of a coenzyme. In one embodiment, the aldehyde dehydrogenase is NAD+ dependent. In another embodiment, the aldehyde dehydrogenase is NADP+ dependent. In one embodiment, the aldehyde dehydrogenase is an NAD+ dependent aldehyde dehydrogenase from bacteria. In some embodiments, the NAD+ dependent aldehyde dehydrogenase is aldA (NP_415933) from E. coli. In another embodiment, the aldehyde dehydrogenase is a cytosolic NADP+ dependent aldehyde dehydrogenase from yeast. In some embodiments, the NADP+ dependent aldehyde dehydrogenase is ALD6 (YPL061W) from S. cerevisiae. In another embodiment, the aldehyde dehydrogenase is a cytosolic NADP+ dependent aldehyde dehydrogenase from bacteria. In some embodiments, the NADP+ dependent aldehyde dehydrogenase is aldB (NP_418045) from E. coli.

In one embodiment, overexpression of an enzyme to increase intracellular levels of a coenzyme comprises coupling supplementation of a co-substrate and overexpression of the enzyme. In one embodiment, the overexpression of an enzyme coupled with supplementation of a co-substrate of that enzyme increase flux through a biochemical pathway. In one embodiment, an NAD+ or NADP+ dependent alcohol dehydrogenase is expressed with a co-substrate. In certain embodiments, an alcohol dehydrogenase is expressed with an isopropanol co-substrate. In one embodiment, an NAD+ or NADP+ dependent glucose dehydrogenase is expressed with a co-substrate. In certain embodiments, a glucose dehydrogenase is expressed with a glucose co-substrate.

In one embodiment, the expression of a transhydrogenase is increased to interconvert NADH and NADPH. In some embodiments, the transhydrogenase is a pyridine nucleotide transhydrogenase. In some embodiments, the pyridine nucleotide transhydrogenase is from bacteria. In certain embodiments, the pyridine nucleotide transhydrogenase is pntAB (beta subunit: NP_416119 alpha subunit: NP_416120) from E. coli. In some embodiments, the pyridine nucleotide transhydrogenase is from human. In certain embodiments, the pyridine nucleotide transhydrogenase is NNT (NP_036475) from Homo sapiens. In certain embodiments, the pyridine nucleotide transhydrogenase is from Solanum tuberosum. In certain embodiments, the pyridine nucleotide transhydrogenase is from Spinacea oleracea.

In some embodiments, it may be useful to increase the expression of endogenous or exogenous proteins to induce endoplasmic reticulum (ER) membrane proliferation. In some embodiments, the induction of endoplasmic reticulum membrane proliferation can improve production of fatty alcohols, aldehydes, or acetates. In one embodiment, the expression of an inactivated HMG-CoA reductase (hydroxymethylglutaryl-CoA reductase) containing one or more ER facing loops is increased. In certain embodiments, the one or more loops is between transmembrane domains 6 and 7 of an inactivated HMG-CoA reductase. In some embodiments, the inactivated HMG-CoA reductase comprises an inactivated protein or chimera which codes for the first 500 amino acids or a subsequence of the first 500 amino acids of Yarrowia lipolytica YALI0E04807p. In other embodiments, the inactivated HMG-CoA reductase comprises an inactivated protein or chimera which codes for the first 522 amino acids or a subsequence of the first 522 amino acids of HMG1 from Saccharomyces cerevisiae (NP_013636.1). In other embodiments, the inactivated HMG-CoA reductase comprises an inactivated protein or chimera which codes for the first 522 amino acids or a subsequence of the first 522 amino acids of HMG2 from Saccharomyces cerevisiae (NP_013555.1). In some embodiments, the expression of one or more regulatory proteins is increased to improve production of fatty alcohols, aldehydes, or acetates. In certain embodiments, the regulatory protein comprises HAC transcription factor from Saccharomyces cerevisiae (NP_116622.1). In certain embodiments, the regulatory protein comprises HAC1 transcription factor from Yarrowia lipolytica (YALI0B2716p).

Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described a fatty alcohol pathway enzymes. Overexpression of a fatty alcohol pathway enzyme or enzymes can occur, for example, through increased expression of an endogenous gene or genes, or through the expression, or increased expression, of an exogenous gene or genes. Therefore, naturally occurring organisms can be readily modified to generate non-natural, fatty alcohol producing microorganisms through overexpression of one or more nucleic acid molecules encoding a fatty alcohol biosynthetic pathway enzyme. In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the fatty alcohol biosynthetic pathways.

Equipped with the present disclosure, the skilled artisan will be able to readily construct the recombinant microorganisms described herein, as the recombinant microorganisms of the disclosure can be constructed using methods well known in the art as exemplified above to exogenously express at least one nucleic acid encoding a fatty alcohol pathway enzyme in sufficient amounts to produce a fatty alcohol.

Methods for constructing and testing the expression levels of a non-naturally occurring fatty alcohol-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubo et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

A variety of mechanisms known in the art can be used to express, or overexpress, exogenous or endogenous genes. For example, an expression vector or vectors can be constructed to harbor one or more fatty alcohol biosynthetic pathway enzyme encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome.

Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. In some embodiments, the present disclosure teaches the use of the bla (bacterial ampR resistance marker). In some embodiments, the present disclosure teaches use of the URA3 marker. In some embodiments, the present disclosure teaches microorganisms comprising the SUC2 gene to permit fermentation in sucrose media.

Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art.

Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)). Exemplary expression control sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

In various embodiments, an expression control sequence may be operably linked to a polynucleotide sequence. By “operably linked” is meant that a polynucleotide sequence and an expression control sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the expression control sequence(s). Operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. Operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide.

In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C₆-C₂₄fatty alcohol, aldehyde, or acetate.

In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acid into a ω-hydroxyfatty acid. In some such embodiments, the enzymes that catalyze the conversion of a fatty acid into a ω-hydroxyfatty acid are selected from the group consisting of XP_504406, XP_504857, XP_504311, XP_500855, XP_500856, XP500402, XP_500097, XP_501748, XP_500560, XP_501148, XP_501667, XP_500273, BAA02041, CAA39366, CAA39367, BAA02210, BAA02211, BAA02212, BAA02213, BAA02214, AAO73952, AAO73953, AAO73954, AAO73955, AAO73956, AAO73958, AAO73959, AAO73960, AAO73961, AAO73957, XP_002546278, BAM49649, AAB80867, AAB17462, ADL27534, AAU24352, AAA87602, CAA34612, ABM17701, AAA25760, CAB51047, AAC82967, WP_011027348, or homologs thereof.

In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6), Y. lipolytica YALI0A15488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A2130g (ALK12).

In other embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of a fatty acyl-CoA into α,β-enoyl-CoA. In some such embodiments, the enzymes that catalyze the conversion of a fatty acyl-CoA into α,β-enoyl-CoA are selected from the group consisting of CAA04659, CAA04660, CAA04661, CAA04662, CAA04663, CAG79214, AAA34322, AAA34361, AAA34363, CAA29901, BAA04761, AAA34891, AAB08643, CAB15271, BAN55749, CAC44516, ADK16968, AEI37634, WP_000973047, WP_025433422, WP_035184107, WP_026484842, CEL80920, WP_026818657. WP_005293707, WP_005883960, or homologs thereof.

In some embodiments, one or more genes of the microbial host encoding acyl-CoA oxidases are deleted or down-regulated to eliminate or reduce the truncation of desired fatty acyl-CoAs beyond a desired chain-length. Such deletion or down-regulation targets include but are not limited to Y. lipolytica POX1(YALI0E32835g). Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g): S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655. CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).

In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more proteins involved in peroxisome biogenesis. In such embodiments, the one or more proteins involved in peroxisome biogenesis are selected from the group consisting of XP_505754, XP_501986, XP_501311, XP_504845, XP_503326, XP_504029, XP_002549868. XP_002547156, XP_002545227, XP_002547350, XP_002546990, EIW11539, EIW08094, EIW11472, EIW09743, EIW0828, or homologs thereof.

In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for one or more unsaturated fatty acyl-CoA intermediates. In one embodiment, the one or more endogenous enzymes comprise one or more diacylglycerol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more diacylglycerol acyltransferases selected from the group consisting of YALI0E32769g, YALI0D07986g and CTRG_06209, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more glycerolphospholipid acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more glycerolphospholipid acyltransferases selected from the group consisting of YALI0E16797g and CTG_04390, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more acyl-CoA/sterol acyltransferases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more acyl-CoA/sterol acyltransferases selected from the group consisting of YALI0F06578g, CTRG_01764 and CTRG_01765, or homolog thereof.

In some embodiments, one or more genes of the microbial host encoding glycerol-3-phosphate acyl transferases (GPATs), lysophosphatidic acid acyltransferases (LPAATs), glycerolphospholipid acyltransferase (GPLATs) and/or diacylglycerol acyltransferases (DGATs) are deleted or downregulated, and replaced with one or more GPATs, LPAATs, GPLATs, or DGATs which prefer to store short-chain fatty acyl-CoAs. Such deletion or downregulation targets include but are not limited to Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, S. cerevisiae YOR245c, Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.

In some preferred embodiments, one or more genes of the microbial host encoding acylglycerol lipases (mono-, di-, or triacylglycerol lipases) and sterol ester esterases are deleted or downregulated and replaced with one or more acylglycerol lipases which prefer long chain acylglycerol substrates. In some embodiments, the one or more endogenous acylglycerol lipase and/or sterol ester esterase enzymes being deleted or downregulated are selected from Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F0010g, Y. lipolytica YALI0C14520g, Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevsiae YKL094W. S. cerevisiae YLL012W, S. cerevisiae YLR020C, Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328. Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049. Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.

In another embodiment, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that consumes fatty acetate products. In one embodiment, the one or more endogenous enzymes comprise one or more sterol esterases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more sterol esterases selected from the group consisting of YALI0E32035g, YALI0E00528g, CTRG_01138, CTRG_01683 and CTRG_04630, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more triacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more triacylglycerol lipases selected from the group consisting of YALI0D17534g, YALI0F10010g, CTRG_00057 and CTRG_06185, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more monoacylglycerol lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more monoacylglycerol lipases selected from the group consisting of YALI0C14520g, CTRG_03360 and CTRG_05049, or homolog thereof. In another embodiment, the one or more endogenous enzymes comprise one or more extracellular lipases. In the context of a recombinant yeast microorganism, the recombinant yeast microorganism is engineered to delete, disrupt, mutate, and/or reduce the activity of one or more extracellular lipases selected from the group consisting of YALI0A20350g, YALI0D19184g, YALI0B09361g, CTRG_05930, CTRG_04188, CTRG_02799, CTRG_03052 and CTRG_03885, or homolog thereof.

In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that (1) break down fatty acids in the course of beta-oxidation and/or (2) oxidize ω-hydroxy fatty acids to fatty acid aldehyde or to dicarboxylic acid in the course of ω-oxidation. In some embodiments, the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of: (i) one or more endogenous acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6); (ii) one or more endogenous (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4). YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7); and (iii) an endogenous (fatty) alcohol oxidase YALI0B14014g (FAO1).

In some embodiments, the Y. lipolytica microorganism into which biosynthesis pathways for the production of C₆-C₂₄fatty alcohol, fatty aldehyde and/or fatty acetate are introduced is H222 ΔP ΔA ΔF ΔURA3. AP denotes deletion of the acyl-CoA oxidase genes (POX1-6) in Y. lipolytica. AA denotes deletion of the (fatty) alcohol dehydrogenase genes (FADH, ADH1-7) in Y. lipolytica. AF denotes deletion of the (fatty) alcohol oxidase gene (FAO1) in Y. lipolytica. ΔURA3 denotes deletion of the URA3 gene in Y. lipolytica, rendering the yeast a uracil auxotroph. In some embodiments, the Y. lipolytica microorganism into which biosynthesis pathways for the production of C₆-C₂₄fatty alcohol, fatty aldehyde and/or fatty acetate are introduced is H222 ΔP ΔA ΔF. In some embodiments, the Y. lipolytica microorganism into which biosynthesis pathways for the production of C₆-C₂₄fatty alcohol, fatty aldehyde and/or fatty acetate are introduced is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.

A wild type isolate of the yeast Y. lipolytica, preferably of the strain H222, can be used as the starting strain for the construction of strains according to the disclosure. The strain H222 was deposited on 29 Apr. 2013 at the DSMZ (Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH, D-38142 Braunschweig) under the number DSM 27185 according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. A selection marker is required for the use of a strain for further genetic processing. This selection marker can be introduced into the strain in a manner known per se, e.g. in the form of the uracil auxotroph. Alternatively, already known uracil auxotrophic strains can be used, preferably the strain H222-S4 (Mauersberger S, Wang H J, Gaillard in C, Barth G & Nicaud J M (2001) J Bacterial 183: 5102-5109). The respective deletion cassette (e.g. POX1-6, FADH, ADH1-7, FAO1) is obtained by PCR or restriction and transformed into Y. lipolytica H222-S4, which can be produced from Y. lipolytica H222 (Mauers-berger et al. (2001)), according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, New York). The creation of H222 ΔP ΔA ΔF ΔURA3 is described in WO 2015/086684, which is herein incorporated by reference in its entirety. Y. lipolytica strain H222 ΔP ΔA ΔF ΔURA3 is used as the starting microorganism for introduction of desaturases and reductases in the present disclosure (see, for example, Examples 7, 9 and 10).

Chemical Conversion of Product from Microorganism Synthesis

The present disclosure describes chemical conversions that can be used to convert a product synthesized by recombinant microorganism into a down-stream product.

In some embodiments, an unsaturated fatty alcohol, aldehyde, acetate, or carboxylic acid produced by a microorganism can undergo subsequent chemical conversion to produce a pheromone, fragrance, flavor, polymer, or polymer intermediate. Non-limiting examples of chemical transformations include esterification, metathesis, and polymerization.

Unsaturated fatty carboxylic acids can be esterified by methods known in the art. For example, Fischer esterification can be used to covert a fatty carboxylic acid to a corresponding fatty ester. See, e.g., Komura, K. et al., Synthesis 2008, 3407-3410.

Elongation of the carbon chain can be performed by known methods to covert an unsaturated fatty alcohol into an elongated derivative thereof. Olefin metastasis catalysts can be performed to increase the number of carbons on the fatty carbon chain and impart Z or E stereochemistry on the corresponding unsaturated product.

In some embodiments, the metathesis catalyst is a tungsten metathesis catalyst, a molybdenum metathesis catalyst, or a ruthenium metathesis catalyst. In certain embodiments, the metathesis catalyst is a tungsten catalyst or a molybdenum catalyst. The catalysts employed in the present invention generally employ metals which can mediate a particular desired chemical reaction. In general, any transition metal (e.g., having d electrons) can be used to form the catalyst, e.g., a metal selected from one of Groups 3-12 of the periodic table or from the lanthanide series. In some embodiments, the metal is selected from Groups 3-8, or, in some cases, from Groups 4-7. In some embodiments, the metal is selected from Group 6. The term “Group 6” refers to the transition metal group comprising chromium, molybdenum, and tungsten. Additionally, the present invention may also include the formation of heterogeneous catalysts containing forms of these elements (e.g., by immobilizing a metal complex on an insoluble substrate, for example, silica).

In general, any metathesis catalyst stable under the reaction conditions and nonreactive with functional groups on the fatty substrate (e.g., alcohol, ester, carboxylic acid, aldehyde, or acetate) can be used with the present disclosure. Such catalysts are, for example, those described by Grubbs (Grubbs, R. H., “Synthesis of large and small molecules using olefin metathesis catalysts.” PMSE Prepr., 2012), herein incorporated by reference in its entirety. Depending on the desired isomer of the olefin, as cis-selective metathesis catalyst may be used, for example one of those described by Shahane et al. (Shahane, S., et al. ChemCatChem, 2013, 5(12): p. 3436-3459), herein incorporated by reference in its entirety. Catalysts exhibiting cis-selectivity have been described previously (Khan, R K., et al. J Am. Chem. Soc., 2013, 135(28): p. 10258-61; Hartung, J. et al. J. Am. Chem. Soc., 2013, 135(28): p. 10183-5; Rosebrugh, L. E., et al. J. Am. Chem. Soc., 2013, 135(4): p. 1276-9; Marx, V. M., et al. J. Am. Chem. Soc., 2013, 135(1): p. 94-7.; Herbert, M. B., et al. Angew. Chem. Int. Ed. Engl., 2013, 52(1): p. 310-4; Keitz, B. K., et al. J. Am. Chem. Soc., 2012, 134(4): p. 2040-3: Keitz, B. K., et al. J. Am. Chem. Soc., 2012, 134(1): p. 693-9; Endo, K. et al. J. Am. Chem. Soc., 2011, 133(22): p. 8525-7).

Additional Z-selective catalysts are described in (Cannon and Grubbs 2013: Bronner et al. 2014; Hartung et al. 2014; Pribisko et al. 2014: Quigley and Grubbs 2014) and are herein incorporated by reference in their entirety. Due to their excellent stability and functional group tolerance, in some embodiments metathesis catalysts include, but are not limited to, neutral ruthenium or osmium metal carbene complexes that possess metal centers that are formally in the +2 oxidation state, have an electron count of 16, are penta-coordinated, and are of the general formula LL′AA′M=CRbRc or LL′AA′M=(C═)nCRbRc (Pederson and Grubbs 2002); wherein

M is ruthenium or osmium;

L and L′ are each independently any neutral electron donor ligand and selected from phosphine, sulfonated phosphine, phosphite, phosphinite, phosphonite, arsine, stibnite, ether, amine, amide, imine, sulfoxide, carboxyl, nitrosyl, pyridine, thioether, or heterocyclic carbenes; and

A and A′ are anionic ligands independently selected from halogen, hydrogen, C1-C20 alkyl, aryl, C1-C20 alkoxide, aryloxide, C2-C20 alkoxycarbonyl, arylcarboxylate, C1-C20 carboxylate, arylsulfonyl, C1-C20 alkylsulfonyl, C1-C20 alkylsulfinyl; each ligand optionally being substituted with C1-C5 alkyl, halogen, C1-C5 alkoxy; or with a phenyl group that is optionally substituted with halogen, C1-C5 alkyl, or C1-C5 alkoxy; and A and A′ together may optionally comprise a bidentate ligand; and

Rb and Rc are independently selected from hydrogen, C1-C20 alkyl, aryl, C1-C20 carboxylate, C1-C20 alkoxy, aryloxy, C1-C20 alkoxycarbonyl, C1-C20 alkylthio, C1-C20 alkylsulfonyl and C1-C20 alkylsulfinyl, each of Rb and Rc optionally substituted with C1-C5 alkyl, halogen, C1-C5 alkoxy or with a phenyl group that is optionally substituted with halogen, C1-C5 alkyl, or C1-C5 alkoxy.

Other metathesis catalysts such as “well defined catalysts” can also be used. Such catalysts include, but are not limited to, Schrock's molybdenum metathesis catalyst, 2,6-diisopropylphenylimido neophylidenemolybdenum (VI) bis(hexafluoro-t-butoxide), described by Grubbs et al. (Tetrahedron 1998, 54: 4413-4450) and Basset's tungsten metathesis catalyst described by Couturier, J. L. et al. (Angew. Chem. Int. Ed. Engl. 1992, 31: 628).

Catalysts useful in the methods of the disclosure also include those described by U.S. Pat. No. 9,776,179, Peryshkov, et al. J. Am. Chem. Soc. 2011, 133: 20754-20757; Wang, et al. Angewandte Chemie, 2013, 52: 1939-1943; Yu, et al. J. Am. Chem. Soc., 2012, 134: 2788-2799: Halford. Chem. Eng. News. 2011, 89 (45): 11; Yu, et al. Nature, 2011, 479: 88-93; Lee. Nature. 2011, 471: 452-453; Meek, et al. Nature, 2011: 471, 461-466; Flook, et al. J. Am. Chem. Soc. 2011, 133: 1784-1786; Zhao, et al. Org Lett., 2011, 13(4): 784-787; Ondi, et al. “High activity, stabilized formulations, efficient synthesis and industrial use of Mo- and W-based metathesis catalysts” XiMo Technology Updates. 2015: http://www.ximo-inc.com/files/ximo/uploads/download/Summary_3.11.15.pdf; Schrock, et al. Macromolecules, 2010: 43, 7515-7522; Peryshkov, et al. Organometallics 2013: 32, 5256-5259; Gerber, et al. Organometallics 2013: 32, 5573-5580; Marinescu, et al. Organometallics 2012: 31, 6336-6343; Wang, et al. Angew. Chem. Int. Ed. 2013: 52, 1939-1943; Wang, et al. Chem. Eur. J. 2013: 19, 2726-2740; and Townsend et al. J. Am. Chem. Soc. 2012: 134, 11334-11337.

Catalysts useful in the methods of the disclosure also include those described in International Pub. No. WO 2014/155185; International Pub. No. WO 2014/172534; U.S. Pat. Appl. Pub. No. 2014/0330018; International Pub. No. WO 2015/003815; and International Pub. No. WO 2015/003814.

Catalysts useful in the methods of the disclosure also include those described in U.S. Pat. Nos. 4,231,947; 4,245,131; 4,427,595; 4,681,956; 4,727,215; International Pub. No. WO 1991/009825; U.S. Pat. Nos. 5,087,710; 5,142,073; 5,146,033; International Pub. No. WO 1992/019631; U.S. Pat. Nos. 6,121,473; 6,346,652; 8,987,531: U.S. Pat. Appl. Pub. No. 2008/0119678; International Pub. No. WO 2008/066754; International Pub. No. WO 2009/094201: U.S. Pat. Appl. Pub. No. 2011/0015430: U.S. Pat. Appl. Pub. No. 2011/0065915; U.S. Pat. Appl. Pub. No. 2011/0077421; International Pub. No. WO 2011/040963; International Pub. No. WO 2011/097642; U.S. Pat. Appl. Pub. No. 2011/0237815; U.S. Pat. Appl. Pub. No. 2012/0302710; International Pub. No. WO 2012/167171: U.S. Pat. Appl. Pub. No. 2012/0323000: U.S. Pat. Appl. Pub. No. 2013/0116434; International Pub. No. WO 2013/070725; U.S. Pat. Appl. Pub. No. 2013/0274482; U.S. Pat. Appl. Pub. No. 2013/0281706; International Pub. No. WO 2014/139679; International Pub. No. WO 2014/169014; U.S. Pat. Appl. Pub. No. 2014/0330018; and U.S. Pat. Appl. Pub. No. 2014/0378637.

Catalysts useful in the methods of the disclosure also include those described in International Pub. No. WO 2007/075427; U.S. Pat. Appl. Pub. No. 2007/0282148; International Pub. No. WO 2009/126831; International Pub. No. WO 2011/069134; U.S. Pat. Appl. Pub. No. 2012/0123133; U.S. Pat. Appl. Pub. No. 2013/0261312: U.S. Pat. Appl. Pub. No. 2013/0296511; International Pub. No. WO 2014/134333; and U.S. Pat. Appl. Pub. No. 2015/0018557.

Catalysts useful in the methods of the disclosure also include those described in U.S. Pat. Appl. Pub. No. 2008/0009598: U.S. Pat. Appl. Pub. No. 2008/0207911: U.S. Pat. Appl. Pub. No. 2008/0275247; U.S. Pat. Appl. Pub. No. 2011/0040099; U.S. Pat. Appl. Pub. No. 2011/0282068; and U.S. Pat. Appl. Pub. No. 2015/0038723.

Catalysts useful in the methods of the disclosure include those described in International Pub. No. WO 2007/140954: U.S. Pat. Appl. Pub. No. 2008/0221345; International Pub. No. WO 2010/037550: U.S. Pat. Appl. Pub. No. 2010/0087644; U.S. Pat. Appl. Pub. No. 2010/0113795; U.S. Pat. Appl. Pub. No. 2010/0174068; International Pub. No. WO 2011/091980; International Pub. No. WO 2012/168183: U.S. Pat. Appl. Pub. No. 2013/0079515; U.S. Pat. Appl. Pub. No. 2013/0144060: U.S. Pat. Appl. Pub. No. 2013/0211096; International Pub. No. WO 2013/135776; International Pub. No. WO 2014/001291; International Pub. No. WO 2014/067767; U.S. Pat. Appl. Pub. No. 2014/0171607; and U.S. Pat. Appl. Pub. No. 2015/0045558.

The catalyst is typically provided in the reaction mixture in a sub-stoichiometric amount (e.g., catalytic amount). In certain embodiments, that amount is in the range of about 0.001 to about 50 mol % with respect to the limiting reagent of the chemical reaction, depending upon which reagent is in stoichiometric excess. In some embodiments, the catalyst is present in less than or equal to about 40 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than or equal to about 30 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than about 20 mol %, less than about 10 mol %, less than about 5 mol %, less than about 2.5 mol %, less than about 1 mol %, less than about 0.5 mol %, less than about 0.1 mol %, less than about 0.015 mol %, less than about 0.01 mol %, less than about 0.0015 mol %, or less, relative to the limiting reagent. In some embodiments, the catalyst is present in the range of about 2.5 mol % to about 5 mol %, relative to the limiting reagent. In some embodiments, the reaction mixture contains about 0.5 mol % catalyst. In the case where the molecular formula of the catalyst complex includes more than one metal, the amount of the catalyst complex used in the reaction may be adjusted accordingly.

In some cases, the methods described herein can be performed in the absence of solvent (e.g., neat). In some cases, the methods can include the use of one or more solvents. Examples of solvents that may be suitable for use in the disclosure include, but are not limited to, benzene, p-cresol, toluene, xylene, diethyl ether, glycol, diethyl ether, petroleum ether, hexane, cyclohexane, pentane, methylene chloride, chloroform, carbon tetrachloride, dioxane, tetrahydrofuran (THF), dimethyl sulfoxide, dimethylformamide, hexamethyl-phosphoric triamide, ethyl acetate, pyridine, triethylamine, picoline, and the like, as well as mixtures thereof. In some embodiments, the solvent is selected from benzene, toluene, pentane, methylene chloride, and THF. In certain embodiments, the solvent is benzene.

In some embodiments, the method is performed under reduced pressure. This may be advantageous in cases where a volatile byproduct, such as ethylene, may be produced during the course of the metathesis reaction. For example, removal of the ethylene byproduct from the reaction vessel may advantageously shift the equilibrium of the metathesis reaction towards formation of the desired product. In some embodiments, the method is performed at a pressure of about less than 760 torr. In some embodiments, the method is performed at a pressure of about less than 700 torr. In some embodiments, the method is performed at a pressure of about less than 650 torr. In some embodiments, the method is performed at a pressure of about less than 600 torr. In some embodiments, the method is performed at a pressure of about less than 550 torr. In some embodiments, the method is performed at a pressure of about less than 500 torr. In some embodiments, the method is performed at a pressure of about less than 450 torr. In some embodiments, the method is performed at a pressure of about less than 400 torr. In some embodiments, the method is performed at a pressure of about less than 350 torr. In some embodiments, the method is performed at a pressure of about less than 300 torr. In some embodiments, the method is performed at a pressure of about less than 250 torr. In some embodiments, the method is performed at a pressure of about less than 200 torr. In some embodiments, the method is performed at a pressure of about less than 150 torr. In some embodiments, the method is performed at a pressure of about less than 100 torr. In some embodiments, the method is performed at a pressure of about less than 90 torr. In some embodiments, the method is performed at a pressure of about less than 80 torr. In some embodiments, the method is performed at a pressure of about less than 70 torr. In some embodiments, the method is performed at a pressure of about less than 60 torr. In some embodiments, the method is performed at a pressure of about less than 50 torr. In some embodiments, the method is performed at a pressure of about less than 40 torr. In some embodiments, the method is performed at a pressure of about less than 30 torr. In some embodiments, the method is performed at a pressure of about less than 20 torr. In some embodiments, the method is performed at a pressure of about 20 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 1 torr. In some embodiments, the method is performed at a pressure of less than about 1 torr.

In some embodiments, the two metathesis reactants are present in equimolar amounts. In some embodiments, the two metathesis reactants are not present in equimolar amounts. In certain embodiments, the two reactants are present in a molar ratio of about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In certain embodiments, the two reactants are present in a molar ratio of about 10:1. In certain embodiments, the two reactants are present in a molar ratio of about 7:1. In certain embodiments, the two reactants are present in a molar ratio of about 5:1. In certain embodiments, the two reactants are present in a molar ratio of about 2:1. In certain embodiments, the two reactants are present in a molar ratio of about 1:10. In certain embodiments, the two reactants are present in a molar ratio of about 1:7. In certain embodiments, the two reactants are present in a molar ratio of about 1:5. In certain embodiments, the two reactants are present in a molar ratio of about 1:2.

In general, the reactions with many of the metathesis catalysts disclosed herein provide yields better than 15%, better than 50%, better than 75%, or better than 90%. In addition, the reactants and products are chosen to provide at least a 5° C. difference, a greater than 20° C. difference, or a greater than 40° C. difference in boiling points. Additionally, the use of metathesis catalysts allows for much faster product formation than byproduct, it is desirable to run these reactions as quickly as practical. In particular, the reactions are performed in less than about 24 hours, less than 12 hours, less than 8 hours, or less than 4 hours.

One of skill in the art will appreciate that the time, temperature and solvent can depend on each other, and that changing one can require changing the others to prepare the pyrethroid products and intermediates in the methods of the disclosure. The metathesis steps can proceed at a variety of temperatures and times. In general, reactions in the methods of the disclosure are conducted using reaction times of several minutes to several days. For example, reaction times of from about 12 hours to about 7 days can be used. In some embodiments, reaction times of 1-5 days can be used. In some embodiments, reaction times of from about 10 minutes to about 10 hours can be used. In general, reactions in the methods of the disclosure are conducted at a temperature of from about 0° C. to about 200° C. For example, reactions can be conducted at 15-100° C. In some embodiments, reaction can be conducted at 20-80° C. In some embodiments, reactions can be conducted at 100-150° C.

Unsaturated fatty esters can be reduced using a suitable reducing agent which selectively reduces the ester to the corresponding aldehyde or alcohol but does not reduce the double bond. An unsaturated fatty ester can be reduced to the corresponding unsaturated fatty aldehyde using di-isobutyl aluminum halide (DIBAL) or Vitride®. The unsaturated fatty aldehyde can be reduced to the corresponding fatty alcohol with, e.g., DIBAL or Vitride®. In some embodiments, the unsaturated fatty ester can be reduced to the corresponding fatty alcohol using AlH₃or 9-Borabicyclo(3.3.1)nonane (9-BBN). (See Galatis, P. Encyclopedia of Reagents for Organic Synthesis. 2001. New York: John Wiley & Sons; and Carey & Sunderburg. Organic Chemistry. Part B: Reactions and Synthesis, 5^thedition, 2007. New York. Springer Sciences)

Pheromone Compositions and Uses Thereof

As described above, products made via the methods described herein are pheromones. Pheromones prepared according to the methods of the invention can be formulated for use as insect control compositions. The pheromone compositions can include a carrier, and/or be contained in a dispenser. The carrier can be, but is not limited to, an inert liquid or solid.

Examples of solid carriers include but are not limited to fillers such as kaolin, bentonite, dolomite, calcium carbonate, talc, powdered magnesia. Fuller's earth, wax, gypsum, diatomaceous earth, rubber, plastic, China clay, mineral earths such as silicas, silica gels, silicates, attaclay, limestone, chalk, loess, clay, dolomite, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal meals, tree bark meal, wood meal and nutshell meal, cellulose powders, attapulgites, montmorillonites, mica, vermiculites, synthetic silicas and synthetic calcium silicates, or compositions of these.

Examples of liquid carriers include, but are not limited to, water; alcohols, such as ethanol, butanol or glycol, as well as their ethers or esters, such as methylglycol acetate; ketones, such as acetone, cyclohexanone, methylethyl ketone, methylisobutylketone, or isophorone: alkanes such as hexane, pentane, or heptanes; aromatic hydrocarbons, such as xylenes or alkyl naphthalenes: mineral or vegetable oils; aliphatic chlorinated hydrocarbons, such as trichloroethane or methylene chloride: aromatic chlorinated hydrocarbons, such as chlorobenzenes; water-soluble or strongly polar solvents such as dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone; liquefied gases: waxes, such as beeswax, lanolin, shellac wax, carnauba wax, fruit wax (such as bayberry or sugar cane wax) candelilla wax, other waxes such as microcrystalline, ozocerite, ceresin, or montan; salts such as monoethanolamine salt, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammonium carbamate and mixtures thereof. Baits or feeding stimulants can also be added to the carrier.

Synergist

In some embodiments, the pheromone composition is combined with an active chemical agent such that a synergistic effect results. The synergistic effect obtained by the taught methods can be quantified according to Colby's formula (i.e. (E)=X+Y−(X*Y/100). See Colby, R. S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations”, 1967 Weeds, vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, by “synergistic” is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount. The pheromone compositions and adjuvants of the present methods can synergistically increase the effectiveness of agricultural active compounds and also agricultural auxiliary compounds.

Thus, in some embodiments, a pheromone composition can be formulated with a synergist. The term, “synergist,” as used herein, refers to a substance that can be used with a pheromone for reducing the amount of the pheromone dose or enhancing the effectiveness of the pheromone for attracting at least one species of insect. The synergist may or may not be an independent attractant of an insect in the absence of a pheromone.

In some embodiments, the synergist is a volatile phytochemical that attracts at least one species of Lepidoptera. The term, “phytochemical,” as used herein, means a compound occurring naturally in a plant species. In a particular embodiment, the synergist is selected from the group comprising β-caryophyllene, iso-caryophyllene, α-humulene, inalool, Z3-hexenol/yl acetate, β-farnesene, benzaldehyde, phenylacetaldehyde, and combinations thereof.

The pheromone composition can contain the pheromone and the synergist in a mixed or otherwise combined form, or it may contain the pheromone and the synergist independently in a non-mixed form.

Insecticide

The pheromone composition can include one or more insecticides. In one embodiment, the insecticides are chemical insecticides known to one skilled in the art. Examples of the chemical insecticides include one or more of pyrethoroid or organophosphorus insecticides, including but are not limited to, cyfluthrin, permethrin, cypermethrin, bifinthrin, fenvalerate, flucythrinate, azinphosmethyl, methyl parathion, buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran, clothianidin, acephate, malathion, quinolphos, chloropyriphos, profenophos, bendiocarb, bifenthrin, chlorpyrifos, cyfluthrin, diazinon, pyrethrum, fenpropathrin, kinoprene, insecticidal soap or oil, neonicotinoids, diamides, avermectin and derivatives, spinosad and derivatives, azadirachtin, pyridalyl, and mixtures thereof.

In another embodiment, the insecticides are one or more biological insecticides known to one skilled in the art. Examples of the biological insecticides include, but are not limited to, azadirachtin (neem oil), toxins from natural pyrethrins, Bacillus thuringiencis and Beauveria bassiana, viruses (e.g., CYD-X™, CYD-X HP™, Germstar™, Madex HP™ and Spod-X™), peptides (Spear-™, Spear-P™, and Spear-C™).

In another embodiment, the insecticides are insecticides that target the nerve and muscle. Examples include acetylcholinesterase (AChE) inhibitors, such as carbamates (e.g., methomyl and thiodicarb) and organophosphates (e.g., chlorpyrifos) GABA-gated chloride channel antagonists, such as cyclodiene organochlorines (e.g., endosulfan) and phenylpyrazoles (e.g., fipronil), sodium channel modulators, such as pyrethrins and pyrethroids (e.g., cypermethrin and λ-cyhalothrin), nicotinic acetylcholine receptor (nAChR) agonists, such as neonicotinoids (e.g., acetamiprid, tiacloprid, thiamethoxam), nicotinic acetylcholine receptor (nAChR) allosteric modulators, such as spinosyns (e.g., spinose and spinetoram), chloride channel activators, such as avermectins and milbemycins (e.g., abamectin, emamectin benzoate), Nicotinic acetylcholine receptor (nAChR) blockers, such as bensultap and cartap, voltage dependent sodium channel blockers, such as indoxacarb and metaflumizone, ryanodine receptor modulator, such as diamides (e.g. dhlorantraniliprole and flubendiamide). In another embodiment, the insecticides are insecticides that target respiration. Examples include chemicals that uncouple oxidative phosphorylation via disruption of the proton gradient, such as chlorfenapyr, and mitochondrial complex I electron transport inhibitors.

In another embodiment, the insecticides are insecticides that target midgut. Examples include microbial disruptors of insect midgut membranes, such as Bacillus thuringiensis and Bacillus sphaericus.

In another embodiment, the insecticides are insecticides that target growth and development. Examples include juvenile hormone mimics, such as juvenile hormone analogues (e.g. fenoxycarb), inhibitors of chitin biosynthesis, Type 0, such as benzoylureas (e.g., flufenoxuron, lufenuron, and novaluron), and ecdysone receptor agonists, such as diacylhydrazines (e.g., methoxyfenozide and tebufenozide)

Stabilizer

According to another embodiment of the disclosure, the pheromone composition may include one or more additives that enhance the stability of the composition. Examples of additives include, but are not limited to, fatty acids and vegetable oils, such as for example olive oil, soybean oil, corn oil, safflower oil, canola oil, and combinations thereof.

Filler

According to another embodiment of the disclosure, the pheromone composition may include one or more fillers. Examples of fillers include, but are not limited to, one or more mineral clays (e.g., attapulgite). In some embodiments, the attractant-composition may include one or more organic thickeners. Examples of such thickeners include, but are not limited to, methyl cellulose, ethyl cellulose, and any combinations thereof.

Solvent

According to another embodiment, the pheromone compositions of the present disclosure can include one or more solvents. Compositions containing solvents are desirable when a user is to employ liquid compositions which may be applied by brushing, dipping, rolling, spraying, or otherwise applying the liquid compositions to substrates on which the user wishes to provide a pheromone coating (e.g., a lure). In some embodiments, the solvent(s) to be used is/are selected so as to solubilize, or substantially solubilize, the one or more ingredients of the pheromone composition. Examples of solvents include, but are not limited to, water, aqueous solvent (e.g., mixture of water and ethanol), ethanol, methanol, chlorinated hydrocarbons, petroleum solvents, turpentine, xvylene, and any combinations thereof.

In some embodiments, the pheromone compositions of the present disclosure comprise organic solvents. Organic solvents are used mainly in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent granular formulations. Sometimes mixtures of solvents are used. In some embodiments, the present disclosure teaches the use of solvents including aliphatic paraffinic oils such as kerosene or refined paraffins. In other embodiments, the present disclosure teaches the use of aromatic solvents such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons are useful as co-solvents to prevent crystallization when the formulation is emulsified into water. Alcohols are sometimes used as co-solvents to increase solvent power.

Solubilizing Agent

In some embodiments, the pheromone compositions of the present disclosure comprise solubilizing agents. A solubilizing agent is a surfactant, which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials inside the hydrophobic part of the micelle. The types of surfactants usually used for solubilization are non-ionics: sorbitan monooleates; sorbitan monooleate ethoxylates; and methyl oleate esters.

Binder

According to another embodiment of the disclosure, the pheromone composition may include one or more binders. Binders can be used to promote association of the pheromone composition with the surface of the material on which said composition is coated. In some embodiments, the binder can be used to promote association of another additive (e.g., insecticide, insect growth regulators, and the like) to the pheromone composition and/or the surface of a material. For example, a binder can include a synthetic or natural resin typically used in paints and coatings. These may be modified to cause the coated surface to be friable enough to allow insects to bite off and ingest the components of the composition (e.g., insecticide, insect growth regulators, and the like), while still maintaining the structural integrity of the coating.

Non-limiting examples of binders include polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose, starch, vinylpyrrolidone/vinyl acetate copolymers and polyvinyl acetate, or compositions of these; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or compositions of these; antifoams such as silicone emulsions, long-chain alcohols, phosphoric esters, acetylene diols, fatty acids or organofluorine compounds, and complexing agents such as: salts of ethylenediaminetetraacetic acid (EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoric acids, or compositions of these.

In some embodiments, the binder also acts a filler and/or a thickener. Examples of such binders include, but are not limited to, one or more of shellac, acrylics, epoxies, alkyds, polyurethanes, linseed oil, tung oil, and any combinations thereof.

Surface-Active Agents

In some embodiments, the pheromone compositions comprise surface-active agents. In some embodiments, the surface-active agents are added to liquid agricultural compositions. In other embodiments, the surface-active agents are added to solid formulations, especially those designed to be diluted with a carrier before application. Thus, in some embodiments, the pheromone compositions comprise surfactants. Surfactants are sometimes used, either alone or with other additives, such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the pheromone on the target. The surface-active agents can be anionic, cationic, or nonionic in character, and can be employed as emulsifying agents, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactants are non-ionics such as: alkyl ethoxylates, linear aliphatic alcohol ethoxylates, and aliphatic amine ethoxylates. Surfactants conventionally used in the art of formulation and which may also be used in the present formulations are described, in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, N.J., 1998, and in Encyclopedia of Surfactants, Vol. I-III. Chemical Publishing Co., New York, 1980-81. In some embodiments, the present disclosure teaches the use of surfactants including alkali metal, alkaline earth metal or ammonium salts of aromatic sulfonic acids, for example, ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and of fatty acids of arylsulfonates, of alkyl ethers, of lauryl ethers, of fatty alcohol sulfates and of fatty alcohol glycol ether sulfates, condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or of the naphthalenesulfonic acids with phenol and formaldehyde, condensates of phenol or phenolsulfonic acid with formaldehyde, condensates of phenol with formaldehyde and sodium sulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-, octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylaryl polyether alcohols, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenols, salts of phosphated triarylphenolethoxylates, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignin-sulfite waste liquors or methylcellulose, or compositions of these.

In some embodiments, the present disclosure teaches other suitable surface-active agents, including salts of alkyl sulfates, such as diethanolammonium lauryl sulfate; alkylarylsulfonate salts, such as calcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-C18 ethoxylate; alcohol-alkylene oxide addition products, such as tridecyl alcohol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonate salts, such as sodium dibutyl-naphthalenesulfonate; dialkyl esters of sulfosuccinate salts, such as sodium di(2-ethylhexyl)sulfosuccinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as lauryl trimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of mono and dialkyl phosphate esters; vegetable oils such as soybean oil, rapeseed/canola oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil and the like; and esters of the above vegetable oils, particularly methyl esters.

Wetting Agents

In some embodiments, the pheromone compositions comprise wetting agents. A wetting agent is a substance that when added to a liquid increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank or other vessel to reduce the wetting time of wettable powders and to improve the penetration of water into water-dispersible granules. In some embodiments, examples of wetting agents used in the pheromone compositions of the present disclosure, including wettable powders, suspension concentrates, and water-dispersible granule formulations are: sodium lauryl sulphate; sodium dioctyl sulphosuccinate; alkyl phenol ethoxylates; and aliphatic alcohol ethoxylates.

Dispersing Agent

In some embodiments, the pheromone compositions of the present disclosure comprise dispersing agents. A dispersing agent is a substance which adsorbs onto the surface of particles and helps to preserve the state of dispersion of the particles and prevents them from reaggregating. In some embodiments, dispersing agents are added to pheromone compositions of the present disclosure to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. In some embodiments, dispersing agents are used in wettable powders, suspension concentrates, and water-dispersible granules. Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to re-aggregation of particles. In some embodiments, the most commonly used surfactants are anionic, non-ionic, or mixtures of the two types.

In some embodiments, for wettable powder formulations, the most common dispersing agents are sodium lignosulphonates. In some embodiments, suspension concentrates provide very good adsorption and stabilization using polyelectrolytes, such as sodium naphthalene sulphonate formaldehyde condensates. In some embodiments, tristyrylphenol ethoxylated phosphate esters are also used. In some embodiments, such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates.

Polymeric Surfactant

In some embodiments, the pheromone compositions of the present disclosure comprise polymeric surfactants. In some embodiments, the polymeric surfactants have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a ‘comb’ surfactant. In some embodiments, these high molecular weight polymers can give very good long-term stability to suspension concentrates, because the hydrophobic backbones have many anchoring points onto the particle surfaces. In some embodiments, examples of dispersing agents used in pheromone compositions of the present disclosure are: sodium lignosulphonates; sodium naphthalene sulphonate formaldehyde condensates; tristyrylphenol ethoxylate phosphate esters; aliphatic alcohol ethoxylates; alkyl ethoxylates; EO-PO block copolymers; and graft copolymers.

Emulsifying Agent

In some embodiments, the pheromone compositions of the present disclosure comprise emulsifying agents. An emulsifying agent is a substance, which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent the two liquids would separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkylphenol or aliphatic alcohol with 12 or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzene sulphonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 will normally provide good stable emulsions. In some embodiments, emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant.

Gelling Agent

In some embodiments, the pheromone compositions comprise gelling agents. Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. In some embodiments, the pheromone compositions comprise one or more thickeners including, but not limited to: montmorillonite, e.g. bentonite; magnesium aluminum silicate; and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickening agents. The types of polysaccharides most commonly used are natural extracts of seeds and seaweeds or synthetic derivatives of cellulose. Some embodiments utilize xanthan and some embodiments utilize cellulose. In some embodiments, the present disclosure teaches the use of thickening agents including, but are not limited to: guar gum; locust bean gum; carrageenan; alginates: methyl cellulose: sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). In some embodiments, the present disclosure teaches the use of other types of anti-settling agents such as modified starches, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

Anti-Foam Agent

In some embodiments, the presence of surfactants, which lower interfacial tension, can cause water-based formulations to foam during mixing operations in production and in application through a spray tank. Thus, in some embodiments, in order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before filling into bottles/spray tanks. Generally, there are two types of anti-foam agents, namely silicones and nonsilicones. Silicones are usually aqueous emulsions of dimethyl polysiloxane, while the nonsilicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the anti-foam agent is to displace the surfactant from the air-water interface.

Preservative

In some embodiments, the pheromone compositions comprise a preservative.

Additional Active Agent

According to another embodiment of the disclosure, the pheromone composition may include one or more insect feeding stimulants. Examples of insect feeding stimulants include, but are not limited to, crude cottonseed oil, fatty acid esters of phytol, fatty acid esters of geranyl geraniol, fatty acid esters of other plant alcohols, plant extracts, and combinations thereof.

According to another embodiment of the disclosure, the pheromone composition may include one or more insect growth regulators (“IGRs”). IGRs may be used to alter the growth of the insect and produce deformed insects. Examples of insect growth regulators include, for example, dimilin.

According to another embodiment of the disclosure, the attractant-composition may include one or more insect sterilants that sterilize the trapped insects or otherwise block their reproductive capacity, thereby reducing the population in the following generation. In some situations allowing the sterilized insects to survive and compete with non-trapped insects for mates is more effective than killing them outright.

Sprayable Compositions

In some embodiments, the pheromone compositions disclosed herein can be formulated as a sprayable composition (i.e., a sprayable pheromone composition). An aqueous solvent can be used in the sprayable composition, e.g., water or a mixture of water and an alcohol, glycol, ketone, or other water-miscible solvent. In some embodiments, the water content of such mixture is at least about 10%, at least about 20%, at least about 30%, at least about 40%, 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some embodiments, the sprayable composition is concentrate, i.e. a concentrated suspension of the pheromone, and other additives (e.g., a waxy substance, a stabilizer, and the like) in the aqueous solvent, and can be diluted to the final use concentration by addition of solvent (e.g., water).

In some embodiments, a waxy substance can be used as a carrier for the pheromone and its positional isomer in the sprayable composition. The waxy substance can be, e.g., a biodegradable wax, such as bees wax, carnauba wax and the like, candelilla wax (hydrocarbon wax), montan wax, shellac and similar waxes, saturated or unsaturated fatty acids, such as lauric, palmitic, oleic or stearic acid, fatty acid amides and esters, hydroxylic fatty acid esters, such as hydroxyethyl or hydroxypropyl fatty acid esters, fatty alcohols, and low molecular weight polyesters such as polyalkylene succinates.

In some embodiments, a stabilizer can be used with the sprayable pheromone compositions. The stabilizer can be used to regulate the particle size of concentrate and/or to allow the preparation of a stable suspension of the pheromone composition. In some embodiments, the stabilizer is selected from hydroxylic and/or ethoxylated polymers. Examples include ethylene oxide and propylene oxide copolymer, polyalcohols, including starch, maltodextrin and other soluble carbohydrates or their ethers or esters, cellulose ethers, gelatin, polyacrylic acid and salts and partial esters thereof and the like. In other embodiments, the stabilizer can include polyvinyl alcohols and copolymers thereof, such as partly hydrolyzed polyvinyl acetate. The stabilizer may be used at a level sufficient to regulate particle size and/or to prepare a stable suspension, e.g., between 0.1% and 15% of the aqueous solution.

In some embodiments, a binder can be used with the sprayable pheromone compositions. In some embodiments, the binder can act to further stabilize the dispersion and/or improve the adhesion of the sprayed dispersion to the target locus (e.g., trap, lure, plant, and the like). The binder can be polysaccharide, such as an alginate, cellulose derivative (acetate, alkyl, carboxymethyl, hydroxyalkyl), starch or starch derivative, dextrin, gum (arabic, guar, locust bean, tragacanth, carrageenan, and the like), sucrose, and the like. The binder can also be a non-carbohydrate, water-soluble polymer such as polyvinyl pyrrolidone, or an acidic polymer such as polyacrylic acid or polymethacrylic acid, in acid and/or salt form, or mixtures of such polymers.

Microencapsulated Pheromones

In some embodiments, the pheromone compositions disclosed herein can be formulated as a microencapsulated pheromone, such as disclosed in Ill'lchev, A L et al., J Econ. Entomol. 2006:99(6):2048-54; and Stelinki, L L et al., J. Econ. Entomol. 2007; 100(4):1360-9. Microencapsulated pheromones (MECs) are small droplets of pheromone enclosed within polymer capsules. The capsules control the release rate of the pheromone into the surrounding environment, and are small enough to be applied in the same method as used to spray insecticides. The effective field longevity of the microencapsulated pheromone formulations can range from a few days to slightly more than a week, depending on inter alia climatic conditions, capsule size and chemical properties.

Slow-Release Formulation

Pheromone compositions can be formulated so as to provide slow release into the atmosphere, and/or so as to be protected from degradation following release. For example, the pheromone compositions can be included in carriers such as microcapsules, biodegradable flakes and paraffin wax-based matrices. Alternatively, the pheromone composition can be formulated as a slow release sprayable.

In certain embodiments, the pheromone composition may include one or more polymeric agents known to one skilled in the art. The polymeric agents may control the rate of release of the composition to the environment. In some embodiments, the polymeric attractant-composition is impervious to environmental conditions. The polymeric agent may also be a sustained-release agent that enables the composition to be released to the environment in a sustained manner.

Examples of polymeric agents include, but are not limited to, celluloses, proteins such as casein, fluorocarbon-based polymers, hydrogenated rosins, lignins, melamine, polyurethanes, vinyl polymers such as polyvinyl acetate (PVAC), polycarbonates, polyvinylidene dinitrile, polyamides, polyvinyl alcohol (PVA), polyamide-aldehyde, polyvinyl aldehyde, polyesters, polyvinyl chloride (PVC), polyethylenes, polystyrenes, polyvinylidene, silicones, and combinations thereof. Examples of celluloses include, but are not limited to, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate-butyrate, cellulose acetate-propionate, cellulose propionate, and combinations thereof.

Other agents which can be used in slow-release or sustained-release formulations include fatty acid esters (such as a sebacate, laurate, palmitate, stearate or arachidate ester) or a fatty alcohols (such as undecanol, dodecanol, tridecanol, tridecenol, tetradecanol, tetradecenol, tetradecadienol, pentadecanol, pentadecenol, hexadecanol, hexadecenol, hexadecadienol, octadecenol and octadecadienol).

Pheromones prepared according to the methods of the invention, as well as compositions containing the pheromones, can be used to control the behavior and/or growth of insects in various environments. The pheromones can be used, for example, to attract or repel male or female insects to or from a particular target area. The pheromones can be used to attract insects away from vulnerable crop areas. The pheromones can also be used example to attract insects as part of a strategy for insect monitoring, mass trapping, lure/attract-and-kill or mating disruption.

Lures

The pheromone compositions of the present disclosure may be coated on or sprayed on a lure, or the lure may be otherwise impregnated with a pheromone composition.

Traps

The pheromone compositions of the disclosure may be used in traps, such as those commonly used to attract any insect species, e.g., insects of the order Lepidoptera. Such traps are well known to one skilled in the art, and are commonly used in many states and countries in insect eradication programs. In one embodiment, the trap includes one or more septa, containers, or storage receptacles for holding the pheromone composition. Thus, in some embodiments, the present disclosure provides a trap loaded with at least one pheromone composition. Thus, the pheromone compositions of the present disclosure can be used in traps for example to attract insects as part of a strategy for insect monitoring, mass trapping, mating disruption, or lure/attract and kill for example by incorporating a toxic substance into the trap to kill insects caught.

Mass trapping involves placing a high density of traps in a crop to be protected so that a high proportion of the insects are removed before the crop is damaged. Lure/attract-and-kill techniques are similar except once the insect is attracted to a lure, it is subjected to a killing agent. Where the killing agent is an insecticide, a dispenser can also contain a bait or feeding stimulant that will entice the insects to ingest an effective amount of an insecticide. The insecticide may be an insecticide known to one skilled in the art. The insecticide may be mixed with the attractant-composition or may be separately present in a trap. Mixtures may perform the dual function of attracting and killing the insect.

Such traps may take any suitable form, and killing traps need not necessarily incorporate toxic substances, the insects being optionally killed by other means, such as drowning or electrocution. Alternatively, the traps can contaminate the insect with a fungus or virus that kills the insect later. Even where the insects are not killed, the trap can serve to remove the male insects from the locale of the female insects, to prevent breeding.

It will be appreciated by a person skilled in the art that a variety of different traps are possible. Suitable examples of such traps include water traps, sticky traps, and one-way traps. Sticky traps come in many varieties. One example of a sticky trap is of cardboard construction, triangular or wedge-shaped in cross-section, where the interior surfaces are coated with a non-drying sticky substance. The insects contact the sticky surface and are caught. Water traps include pans of water and detergent that are used to trap insects. The detergent destroys the surface tension of the water, causing insects that are attracted to the pan, to drown in the water. One-way traps allow an insect to enter the trap but prevent it from exiting. The traps of the disclosure can be colored brightly, to provide additional attraction for the insects.

In some embodiments, the pheromone traps containing the composition may be combined with other kinds of trapping mechanisms. For example, in addition to the pheromone composition, the trap may include one or more florescent lights, one or more sticky substrates and/or one or more colored surfaces for attracting moths. In other embodiments, the pheromone trap containing the composition may not have other kinds of trapping mechanisms.

The trap may be set at any time of the year in a field. Those of skill in the art can readily determine an appropriate amount of the compositions to use in a particular trap, and can also determine an appropriate density of traps/acre of crop field to be protected.

The trap can be positioned in an area infested (or potentially infested) with insects. Generally, the trap is placed on or close to a tree or plant. The aroma of the pheromone attracts the insects to the trap. The insects can then be caught, immobilized and/or killed within the trap, for example, by the killing agent present in the trap.

Traps may also be placed within an orchard to overwhelm the pheromones emitted by the females, so that the males simply cannot locate the females. In this respect, a trap need be nothing more than a simple apparatus, for example, a protected wickable to dispense pheromone.

The traps of the present disclosure may be provided in made-up form, where the compound of the disclosure has already been applied. In such an instance, depending on the half-life of the compound, the compound may be exposed, or may be sealed in conventional manner, such as is standard with other aromatic dispensers, the seal only being removed once the trap is in place.

Alternatively, the traps may be sold separately, and the compound of the disclosure provided in dispensable format so that an amount may be applied to trap, once the trap is in place. Thus, the present disclosure may provide the compound in a sachet or other dispenser.

Dispenser

Pheromone compositions can be used in conjunction with a dispenser for release of the composition in a particular environment. Any suitable dispenser known in the art can be used. Examples of such dispensers include but are not limited to, aerosol emitters, hand-applied dispensers, bubble caps comprising a reservoir with a permeable barrier through which pheromones are slowly released, pads, beads, tubes rods, spirals or balls composed of rubber, plastic, leather, cotton, cotton wool, wood or wood products that are impregnated with the pheromone composition. For example, polyvinyl chloride laminates, pellets, granules, ropes or spirals from which the pheromone composition evaporates, or rubber septa. One of skill in the art will be able to select suitable carriers and/or dispensers for the desired mode of application, storage, transport or handling.

In another embodiment, a device may be used that contaminates the male insects with a powder containing the pheromone substance itself. The contaminated males then fly off and provide a source of mating disruption by permeating the atmosphere with the pheromone substance, or by attracting other males to the contaminated males, rather than to real females.

Behavior Modification

Pheromone compositions prepared according to the methods disclosed herein can be used to control or modulate the behavior of insects. In some embodiments, the behavior of the target insect can be modulated in a tunable manner inter alia by varying the ratio of the pheromone to the positional isomer in the composition such that the insect is attracted to a particular locus but does not contact said locus or such the insect in fact contacts said locus. Thus, in some embodiments, the pheromones can be used to attract insects away from vulnerable crop areas. Accordingly, the disclosure also provides a method for attracting insects to a locus. The method includes administering to a locus an effective amount of the pheromone composition.

The method of mating disruption may include periodically monitoring the total number or quantity of the trapped insects. The monitoring may be performed by counting the number of insects trapped for a predetermined period of time such as, for example, daily, Weekly, bi-Weekly, monthly, once-in-three months, or any other time periods selected by the monitor. Such monitoring of the trapped insects may help estimate the population of insects for that particular period, and thereby help determine a particular type and/or dosage of pest control in an integrated pest management system. For example, a discovery of a high insect population can necessitate the use of methods for removal of the insect. Early warning of an infestation in a new habitat can allow action to be taken before the population becomes unmanageable. Conversely, a discovery of a low insect population can lead to a decision that it is sufficient to continue monitoring the population. Insect populations can be monitored regularly so that the insects are only controlled when they reach a certain threshold. This provides cost-effective control of the insects and reduces the environmental impact of the use of insecticides.

Mating Disruption

Pheromones prepared according to the methods of the disclosure can also be used to disrupt mating. Mating disruption is a pest management technique designed to control insect pests by introducing artificial stimuli (e.g., a pheromone composition as disclosed herein) that confuses the insects and disrupts mating localization and/or courtship, thereby preventing mating and blocking the reproductive cycle.

In many insect species of interest to agriculture, such as those in the order Lepidoptera, females emit an airborne trail of a specific chemical blend constituting that species' sex pheromone. This aerial trail is referred to as a pheromone plume. Males of that species use the information contained in the pheromone plume to locate the emitting female (known as a “calling” female). Mating disruption exploits the male insects' natural response to follow the plume by introducing a synthetic pheromone into the insects' habitat, which is designed to mimic the sex pheromone produced by the female insect. Thus, in some embodiments, the synthetic pheromone utilized in mating disruption is a synthetically derived pheromone composition comprising a pheromone having a chemical structure of a sex pheromone and a positional isomer thereof which is not produced by the target insect.

The general effect of mating disruption is to confuse the male insects by masking the natural pheromone plumes, causing the males to follow “false pheromone trails” at the expense of finding mates, and affecting the males' ability to respond to “calling” females. Consequently, the male population experiences a reduced probability of successfully locating and mating with females, which leads to the eventual cessation of breeding and collapse of the insect infestation.

Strategies of mating disruption include confusion, trail-masking and false-trail following. Constant exposure of insects to a high concentration of a pheromone can prevent male insects from responding to normal levels of the pheromone released by female insects. Trail-masking uses a pheromone to destroy the trail of pheromones released by females. False-trail following is carried out by laying numerous spots of a pheromone in high concentration to present the male with many false trails to follow. When released in sufficiently high quantities, the male insects are unable to find the natural source of the sex pheromones (the female insects) so that mating cannot occur.

In some embodiments, a wick or trap may be adapted to emit a pheromone for a period at least equivalent to the breeding season(s) of the midge, thus causing mating disruption. If the midge has an extended breeding season, or repeated breeding season, the present disclosure provides a wick or trap capable of emitting pheromone for a period of time, especially about two weeks, and generally between about 1 and 4 weeks and up to 6 weeks, which may be rotated or replaced by subsequent similar traps. A plurality of traps containing the pheromone composition may be placed in a locus, e.g., adjacent to a crop field. The locations of the traps, and the height of the traps from ground may be selected in accordance with methods known to one skilled in the art.

Alternatively, the pheromone composition may be dispensed from formulations such as microcapsules or twist-ties, such as are commonly used for disruption of the mating of insect pests.

Attract and Kill

The attract and kill method utilizes an attractant, such as a sex pheromone, to lure insects of the target species to an insecticidal chemical, surface, device, etc., for mass-killing and ultimate population suppression, and can have the same effect as mass-trapping. For instance, when a synthetic female sex pheromone is used to lure male pests, e.g., moths, in an attract-and-kill strategy, a large number of male moths must be killed over extended periods of time to reduce matings and reproduction, and ultimately suppress the pest population. The attract-and-kill approach may be a favorable alternative to mass-trapping because no trap-servicing or other frequent maintenance is required. In various embodiments described herein, a recombinant microorganism can co-express (i) a pathway for production of an insect pheromone and (ii) a protein, peptide, oligonucleotide, or small molecule which is toxic to the insect. In this way, the recombinant microorganism can co-produce substances suitable for use in an attract-and-kill approach.

As will be apparent to one of skill in the art, the amount of a pheromone or pheromone composition used for a particular application can vary depending on several factors such as the type and level of infestation; the type of composition used: the concentration of the active components; how the composition is provided, for example, the type of dispenser used; the type of location to be treated; the length of time the method is to be used for; and environmental factors such as temperature, wind speed and direction, rainfall and humidity. Those of skill in the art will be able to determine an effective amount of a pheromone or pheromone composition for use in a given application.

As used herein, an “effective amount” means that amount of the disclosed pheromone composition that is sufficient to affect desired results. An effective amount can be administered in one or more administrations. For example, an effective amount of the composition may refer to an amount of the pheromone composition that is sufficient to attract a given insect to a given locus. Further, an effective amount of the composition may refer to an amount of the pheromone composition that is sufficient to disrupt mating of a particular insect population of interest in a given locality.

EXAMPLES
Example 1. Production of Pheromones Products from Enzymatically-Derived Gondoic Acid through Metathesis and Chemical Conversion

This example illustrates that different fatty acids can be used as a starting material for the biosynthetic production of a pheromone or pheromone precursor. The product obtained from the biosynthetic process disclosed herein can be subject to further chemical conversions to generate different products.

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Enzymatic two carbon elongation of oleic acid yields gondoic acid. After esterification, gondoic fatty acid methyl ester (FAME) can then converted via Z-selective olefin metathesis into C16 and C18 FAME products containing a C11 unsaturation. Upon reduction of the ester, aldehyde and fatty alcohol pheromone materials can be produced. Acetylation of the fatty alcohol product can generate the corresponding fatty acetate pheromones. Additionally, gondoic acid can be directly converted into C20 fatty aldehyde, alcohol and acetate pheromones through application of the same chemical transformation of enzymatically modified oleic acid.

Prophetic Example 2: Tailored Synthetic Blends

This prophetic example illustrates that the recombinant microorganisms disclosed herein can be used to create synthetic blends of insect pheromones.

As shown in the scheme depicted in FIG. 54, using tetradecyl-ACP (14:ACP), a blend of E- and Z-tetradecenyl acetate (E11-14:OAc and Z11-14:OAC) pheromones can be produced with the recombinant microorganism. This blend is produced by a variety of insects, e.g., Chonstoneura roseceana (a moth of the Tortricidae family).

Similarly, using hexadecyl-ACP (16:ACP), a blend of Z- and E hexadecenyl acetate pheromones (E11-16:OAc and Z11-16:OAc) can be produced with the recombinant microorganism.

The microorganism can be engineered with different desaturases, or other enzymes such as reductases, etc. to produce the desired blend of pheromones. One blend of particular relevance capable of being produced using the recombinant microorganisms and methods of the instant invention is a 97:3 ratio of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald).

Example 3: Expression of Transmembrane Alcohol-Forming Reductases in S. cerevisiae

Background and Rationale

Engineering microbial production of insect fatty alcohols from fatty acids entails the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects (as well as some microalgae in the case of fatty alcohol reductase) and can be used to construct the synthetic pathway in yeasts, which are preferred production hosts. A number of transmembrane desaturases and alcohol-forming reductase variants will be screened to identify ensembles which allow high level synthesis of a single insect fatty alcohol or a blend of fatty alcohols. Additionally, these enzymes will be screened across multiple hosts (Saccharomyces cerevisiae, Candida tropicalis, and Yarrowia lipolytica) to optimize the search toward finding a suitable host for optimum expression of these transmembrane proteins.

Summary of Approach

Three alcohol-forming reductases of insect origin were selected.

Nucleic acids encoding the reductases were synthesized (synthons) with codon optimization for expression in S. cerevisiae.

Each nucleic acid encoding a given reductase was subcloned into an episomal expression cassette under the Gall promoter.

S. cerevisiae wild-type and beta-oxidation deletion mutant were transformed with expression constructs.

Heterologous protein was induced by galactose, and functional expression of the reductases was assessed in vivo via bioconversion of Z11-hexadecenoic acid into Z11-hexedecenol.

GC-MS analysis was used to identify and quantify metabolites.

Results

Alcohol-forming reductase variants were screened for activity in S. cerevisiae W303 (wild type) and BY4742 ΔPOX1 (beta-oxidation deletion mutant). Z11-hexadecenoic acid was chosen as a substrate in assessing enzyme activity. The in vivo bioconversion assay showed that the expression of enzyme variants derived from Spodoptera littoralis. Helicoverpa armigera, and Agrotis segetum (Ding, B-J., Löfstedt, C. Analysis of the Agrotis segetum pheromone gland transcriptome in the light of sex pheromone biosynthesis. BMC Genomics 16:711 (2015)) in W303A conferred Z11-hexadecenol production, and reached up-to ˜37 μM (8 mg/L), ˜70 μM (˜16 mg/L), and 11 μM (˜3 mg/L), respectively, within 48h of protein induction (FIG. 5 and FIG. 6). Biologically-produced Z11-hexadecenol matched authentic Z11-hexadecenol standard (Bedoukian) as determined via GC-MS (FIG. 7). BY4742 ΔPOX1 was also explored as an expression host since deletion in the key beta-oxidation pathway enzyme could limit the degradation of Z11-hexadecenoic acid. Expressing the reductase variants in the beta-oxidation deletion mutant, however, reduced the product titer when compared to expression in the wild-type host (FIG. 5). One contributing factor of titer reduction when using BY4742 ΔPOX1 as a host was the reduction of biomass when compared to W303 (FIG. 8).

Therefore, functional expression of at least two alcohol-forming reductases in S. cerevisiae conferred bioconversion of Z11-hexadecenoic acid into Z11-hexedecenol.

Conclusions

Functional expression of insect transmembrane alcohol-forming reductase in S. cerevisiae was demonstrated. Among the reductases tested, the variant derived from Helicoverpa armigera is most active toward Z11-hexadecenoic acid.

The bioconversion of other fatty acid substrates can be explored to assess enzyme plasticity.

Materials & Methods

Strain Construction and Functional Expression Assay

S. cerevisiae W303 (MATA ura3-1 trp1-1 leu2-3_112 his3-11_15 ade2-1 can1-100) and BY4742 (MATa POX1::kanMX his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) were used as expression hosts. DNA sequences which encode fatty alcohol reductase variants were redesigned to optimize expression in S. cerevisiae (SEQ ID NOs: 1-3). Generated synthons (Genscript) were cloned into pESC-URA vector using BamHI-XhoI sites to facilitate protein expression utilizing the Gall promoter. The resulting plasmid constructs were used to transform W303, and positive transformants were selected on CM agar medium (with 2% glucose, and lacking uracil) (Teknova). To assess functional expression, two positive transformation clones that have been patched on CM agar medium (with 2% glucose, and lacking uracil) were used to seed CM liquid medium using a 24 deep-well plate format. To induce protein expression, the overnight cultures that had been grown at 28° C. were then supplemented with galactose, raffinose, and YNB to a final concentration of 2%, 1%, and 6.7 g/L, respectively. Post 24h of protein induction, the bioconversion substrate Z11-hexadecenoic acid (in ethanol) or heptadecanoic acid (in ethanol) was added to a final concentration of 300 mg/L. Bioconversion assay proceeded for 48 h at 28° C. prior to GC-MS analysis.

Metabolite Extraction and GC-MS Detection

The lipids were extracted according to a modified procedure of Hagström et al. (2012) (Hagström, Å. K., Liénard, M. A., Groot, A. T., Hedenström, E. & Löfstedt, C. Semi-Selective Fatty Acyl Reductases from Four Heliothine Moths Influence the Specific Pheromone Composition. PLoS One 7: e37230 (2012)), 1.5 mL-cell culture was transferred to a 15 mL falcon tube. The cell suspension was acidified with 1 mL 5 N HCl, 5 μL tetradecanedioic acid (10 mM in ethanol) was added as internal standard. The mixture was extracted by adding 1.5 mL hexane, then shaken for 1 h at 37° C., 250 rpm. To facilitate phase separation, the sample was centrifuged for 10 min at 2000 g, 1 mL of the organic hexane phase was then transferred to a 1.5 mL plastic tube. The solvent was removed by heating the sample 30 min at 90° C. After the sample was evaporated to dryness, 50 μL of BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide containing 1% of trimethylchlorosilane) was added. The 1.5 mL plastic tubes were shaken vigorously two times for 10 s. Prior to the transfer into a screw cap GC glass vial containing a glass insert, the sample was centrifuged for 1 min (13000 rpm). The vials were capped and heated for 30 min at 90° C. The trimethylsilyl-esters, which were generated by this method were subsequently analyzed by GC-MS analysis. GC-MS parameters are specified in Table 6. The use of SIM mode (characteristic product and IS ions) increases detection sensitivity by reducing background noise, allowing detection of the product as low as 2.4 μM (0.6 mg/L). A further reduction in the split ratio offers the possibility to further increase the sensitivity for future applications. A Z11-hexadecenol calibration curve shown in FIG. 9 was used to quantify the Z11-hexadecenol produced from yeasts. The bioconversion of heptadecanoic acid was also tested since the easily distinguished heptadecanol product could be used to benchmark successful GC-MS runs. However, none of the reductase tested showed any activity toward heptadecanoic acid.

TABLE 6

GC-MS parameters

System
Agilent 6890 N GC, ChemStation G1701EA E.02.01.1177

Column
Rtx-5 30 m × 320 μm × 25 μm

Pressure = 11.74 psi; Flow = 7.1 mL/min

Inlet
Heater = 250° C.; Pressure = 11.74 psi;

Total Flow {He} = 19.5 mL/min

Carrier
He @ 147 cm/sec, 11.74 psi

Signal
Data rate = 2 Hz/0.1 min

Oven
150° C. for 1 min

Ramp 12° C./min to 220° C., hold 3 min

Ramp 35° C./min to 300° C., hold 4 min

Injection
Split, 250° C.

Split ratio −20:1

Detector
HP 5973 MSD in SIM mode (m/z: 297.3 and 387.3),

100 msec Dwell, EMV mode: Gain factor 1,

3 min solvent delay, 8.33 cycles/sec|

Sample
Injection volume = 1 uL

Example 4: Expression of Transmembrane Desaturases in S. cerevisiae

Background and Rationale

Engineering microbial production of insect fatty alcohols from fatty acids requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects as well as some microalgae. A number of transmembrane desaturases and alcohol-forming reductase variants will be screened to identify ensembles which allow high level synthesis of a single insect fatty alcohol or a blend of fatty alcohols. Additionally, these enzymes will be screened across multiple hosts (Saccharomyces cerevisiae, Candida tropicalis, and Yarrowia lipolytica) to optimize the search toward finding a suitable host for optimum expression of these transmembrane proteins.

Summary of Approach

A small set of desaturases (insect origin: Agrotis segetum, Trichoplusia ni, Amyelois transitella, Helicoverpa zea, and marine diatom: Thalassiosira pseudonana) were selected as a test case to explore and establish functional expression assays, metabolite extraction methods, and analytical chemistry.

A synthetic cassette for expression of the desaturases in S. cerevisiae was constructed. The cassette consists of the OLE1 promoter region, OLE1 N-terminal leader sequence, and VSP13 terminator.

The expression cassette was tested for functionality via expression of a GFP variant. Validation of the cassette allowed its utilization for exploring expression of insect desaturase.

S. cerevisiae ΔOLE1 was transformed with expression constructs containing heterologous desaturases. Functionality of the desaturases was assessed via the ability to rescue growth of ΔOLE1 without exogenous supplementation of unsaturated fatty acid (UFA). S. cerevisiae desaturase (OLE1) was used as a positive control of successful complementation.

Functionality of the desaturase was validated via an in vive bioconversion of hexadecanoic acid (palmitic acid) into (Z)-11-hexadecenoic acid (palmitvaccenic acid).

GC-MS analysis was used to identify and quantify metabolites.

Results

Transmembrane desaturase variants were screened in S. cerevisiae. Three variants were initially tested to explore and establish functional expression assays, metabolite extraction methods, and analytical chemistry. To allow functional expression of these desaturases in S. cerevisiae, an episomal synthetic expression cassette termed pOLE1 cassette (FIG. 10) was constructed, which consisted of an OLE1 promoter region, an N-terminal leader sequence encoding for the first 27 amino acids of S. cerevisiae OLE1, and a terminator region of VPS13 (a protein involved in the protospore membrane formation, the terminator of which has been previously characterized to increase heterologous protein expression potentially by extending mRNA half-life). The functionality of the pOLE1 cassette was validated via its ability to express a GFP (FIG. 11A-FIG. 11E). Subsequently, insect desaturase synthons, and yeast OLE synthon were cloned into the pOLE1 cassette, and expressed in S. cerevisiae ΔOLE1 strain. This strain was chosen since deletion of the OLE1 allele (which encodes for palmitoyl:CoA/stearoyl:CoA (z)-9-desaturase) allows its utilization as a tool to screen for functional insect desaturase. Specifically, an active desaturase would allow complementation of growth without requiring exogenous supplementation of UFAs. Expression of OLE1 using pOLE1 cassette complemented growth of ΔOLE1 growth without UFA (FIG. 11A-FIG. 11E); therefore, it serves as a positive control in the complementation assays. When insect desaturases were expressed, we observed that they rescued ΔOLE1 growth without UFA at varying degree. On rich medium (YPD) agar plate, expression of S. cerevisiae OLE1 conferred the highest level of growth, followed by T. ni desaturase (FIG. 12A). The latter indicated that production of unsaturated fatty acyl:CoA by T. ni desaturase could act as a surrogate to the missing (Z)-9-hexadecenoyl:CoA biosynthesis in ΔOLE1. Expression of T. pseudonana and A. segetum desaturases did not appear to rescue growth on YPD very well (FIG. 12A). When patched on minimal medium (CM-Ura glucose) agar plate, only expression of S. cerevisiae OLE1 and T. ni desaturase rescued ΔOLE1 growth without exogenous UFA (FIG. 12B). Expression of T. pseudonana and A. segetum desaturases did not confer growth of ΔOLE1 on minimal medium agar, suggesting their limited activity in producing UFA (results not shown). Screening a desaturase library in Candida tropicalis identified functional expression of A. transitella and H. zea desaturases. When these desaturases were expressed in ΔOLE1, they conferred growth without UFA on both YPD and CM-Ura glucose media similar to expression of T. ni desaturase (FIG. 12B).

Functional expression of the heterologous desaturases was further characterized via in vivo bioconversion of palmitic acid into insect-specific UFA. Post ˜96h-cultivation in minimal medium containing palmitic acid, total fatty acid analysis of S. cerevisiae ΔOLE1 expressing T. ni desaturase revealed production of a new fatty acid species (Z)-11-hexadecenoic acid that is not present in the control strain which expresses native yeast OLE1 desaturase (FIG. 13A-FIG. 13B). (Z)-11-hexadecenoic acid is not detected in strains expressing A. segetum, or T. pseudonana desaturase (results not shown). In addition to (Z)-11-hexadecenoic acid, (Z)-9-hexadecenoic acid was also detected in ΔOLE1 strain expressing T. ni desaturase (FIG. 13A-FIG. 13B). Under the cultivation condition, C16-fatty acid in the ΔOLE1 expressing T. ni desaturase is composed of approximately 84.7% hexadecanoic acid, 5.6% (Z)-9-hexadecenoic acid and 9.8% (Z)-11-hexadeceneoic acid. In comparison, the C16 fatty acid fraction of ΔOLE1 expressing OLE1 desaturase is composed of approximately 68.6% hexadecanoic acid and 31.4% (Z)-9-hexadecenoic acid. (Z)-1 I1-hexadecenoic acid biosynthesis in ΔOLE1 expressing T. ni desaturase account for 1.5 mg/L. The amount of total fatty acids and each fatty acid within this mixture can be quantified. The biologically produced (Z)-11-hexadecenoic acid also match the retention time and fragmentation pattern of authentic standard (Z)-11-hexadecenoic acid (Larodan) as determined by GC-MS (FIG. 14A-FIG. 14B). Therefore, the regio- and stereoisomer of the biologically produced (Z)-11-hexadecenoic acid was confirmed. In vivo characterization of A. transitella and H. zea desaturase can also be done.

In summary, at least three insect desaturases capable of rescuing growth of S. cerevisiae ΔOLE1 without exogenous supplementation of UFA, i.e. (Z)-9-hexadecenoic acid (palmitoleic acid), were identified.

The extent of growth on rich medium (YPD) of S. cerevisiae ΔOLE1 bearing the expression construct was in the following order of desaturase content: OLE1, T. ni. T, pseudonana, and A. segetum.

The extent of growth on minimal medium (CM Glucose w/out uracil) of S. cerevisiae ΔOLE1 bearing the expression construct was in the following order of desaturase content: OLE1. T. ni.

Complementation assays using A. transitella and H. zea desaturases were also done, demonstrating functional expression in Candida tropicalis shown via in vivo bioconversion assay. These desaturases also complemented S. cerevisiae ΔOLE1 growth on rich and minimal media at least as well as T. ni desaturase.

Expression of T. pseudonana and A. segetum desaturases did not confer growth of S. cerevisiae ΔOLE1 on minimal medium without UFAs even after an extended incubation period up to 14 days. No (Z)-11-hexadecenoic acid was observed in strains harboring T. pseudonana or A. segetum desaturase.

Conclusions

Functional expression of transmembrane desaturases of insect origin in S. cerevisiae has been achieved.

The activity of a given heterologous desaturase can be assessed from its ability to complement growth of S. cerevisiae ΔOLE1 without exogenous palmitoleic supplementation, and its ability to convert palmitic acid into insect pheromone precursors (Z)-11-hexadecenoic acid.

Functional expression and/or activity of insect desaturase in S. cerevisiae varies widely depending on sequence origin. Variants derived from T. ni exhibited the best activity compared to A. segetum and T. pseudonana, as measured by the above criteria.

Desaturases derived from A. transitella and H. zea complemented ΔOLE1 as well as T. ni desaturase. Bioconversion assays using these desaturases can be done.

The bioconversion of other fatty acid substrates can be explored to assess enzyme plasticity.

Materials & Methods

Strain Construction and Functional Expression Assay

S. cerevisiae ΔOLE1 (MATA OLE::LEU2 ura3-52 his4) was used as an expression host. A synthetic expression cassette termed pOLE1 (FIG. 10. SEQ ID NO: 4) which comprises the OLE1 promoter region (SEQ ID NOs: 5 and 6), nucleotides encoding for 27 N-terminal amino acids of the OLE1 leader sequence (SEQ ID NO: 7), and a VPS13 terminator sequence (SEQ ID NO: 8) was created, and cloned into pESC-URA vector in between SacI and EcoRI sites. To test the functionality of the pOLE1 cassette, Dasher GFP synthon was inserted in between SpeI and NotI sites to create pOLE1-GFP plasmid. Competent ΔOLE1 was transformed with pOLE1-GFP, and plated on CM-Ura glucose agar plate (Teknova) containing UFA (20 mm CM-URA glucose agar plate was coated with 100 μL CM-Ura glucose medium containing 1% tergitol, and 3 μL palmitoleic acid). After incubation at 30° C. for 5 days, Dasher GFP expression was apparent as displayed by green coloration of ΔOLE1 transformants. This result showed that the pOLE1 cassette was capable of driving heterologous protein expression. Validation of ΔOLE1 complementation was performed by restoring OLE1 activity. Specifically, native S. cerevisiae OLE1 synthon was inserted into pOLE1 cassette devoid of the leader sequence to create pOLE1-OLE1 plasmid. After transformation of ΔOLE1, and selection on CM-Ura glucose agar containing UFA, single colonies were patched onto YPD and CM-Ura glucose without UFA. After incubation at 30° C. for 5 days, growth was observed (FIG. 11A-FIG. 11E). As expected, Dasher GFP expression could not complement ΔOLE1 growth without UFA (FIG. 11A-FIG. 11E). DNA sequences which encode for desaturase variants were synthesized (to include nucleotide changes which remove restriction sites used for cloning purposes), and cloned into pOLE1 using SpeI-NotI sites (Genscript, SEQ ID NOs: 9-13). Complementation assay of ΔOLE1 with insect desaturases were performed in the same way as with OLE1 desaturase.

To assess functional expression, two positive transformation clones that had been patched on CM-Ura glucose agar medium containing UFA were inoculated in 1.5 mL CM-Ura glucose liquid medium containing palmitic acid (in ethanol) at a final concentration of 300 mg/L, and with 6.7 g/L of YNB. For (z)-11-hexadecenoic isomer confirmation, a 20 mL culture was generated. Bioconversion assay proceeded for 96 h at 28° C. prior to GC-MS analysis.

Metabolite Extraction and GC-MS Detection

Total lipid composition as well as the (Z)-11-hexadecenoic acid quantification was based on modified procedures by Moss et al. (1982) (Moss, C. W., Shinoda, T. & Samuels, J. W. Determination of cellular fatty acid compositions of various yeasts by gas-liquid chromatography. J. Clin. Microbiol. 16: 1073-1079 (1982)) and Yousuf et al (2010) (Yousuf, A., Sannino, F., Addorisio, V. & Pirozzi, D. Microbial Conversion of Olive Oil Mill Wastewaters into Lipids Suitable for Biodiesel Production. J. Agric. Food Chem. 58: 8630-8635 (2010)). The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL screw-cap GC-vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM heptadecanoic were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13.000 rpm, afterwards 450 μL of the organic phase were transferred into a new 1.5 mL plastic tube. The aqueous phase was extracted a second time with 500 μL chloroform, this time without heptadecanoic acid. The combined organic phases were evaporated at 90° C. After cooling to room temperature, residual fatty acid methyl esters and free fatty acids were dissolved and derivatized in methanol containing 0.2 M TMSH (trimethylsulfonium hydroxide).

The regioselectivity of biologically produced (Z)-11-hexadecenoic acid was determined by comparing the fragmentation patterns of the dimethyl disulfide (DMDS) derivative with the DMDS derivative of an authentic standard. A yeast culture was split into 12 aliquots (to not change any parameters in the developed procedure). The cells were pelleted, which yielded 63 mg cells (ccw) on average (755 mg from 18 mL culture). The pellets were subjected to base methanolysis as described above. However, after acidification the samples were combined in a 50 mL Falcon tube. The combined sample was extracted two times with 10 mL chloroform. The mixture was centrifuged 10 min at 3000 rpm to achieve a better phase separation. The combined organic phases, which were combined in a new 50 mL Falcon and were washed consecutively with 10 mL brine and 10 mL water. The organic phase was dried with anhydrous sodium sulfate and concentrated in vacuo. The concentrated oil was dissolved in 1.5 mL chloroform and transferred to a 1.5 mL plastic tube. The chloroform was evaporated at 90° C. The remaining sample was the dissolved in 50 μL methyl tert-butyl ether (MTBE). The 50 μL were split into 1, 5, 10 and 20 μL and transferred into GC-vials without insert. To each vial 200 μL DMDS (dimethyl disulfide) and 50 μL MTBE (containing 60 mg/mL iodine) were added. After the mixture was heated 48 h at 50° C., excess iodine was removed by the addition of 100 μL saturated sodium thiosulfate solution. The samples were transferred to plastic vials and extracted to times with 500 μL dichloromethane. The combined organic phases were transferred to a new 1.5 mL plastic vial and evaporated at 90° C. The samples were taken up in 50 μL DCM and transferred to a GC-vial. The sample was analyzed by GC-MS (Table 7) using the method of Hagström et al. (2013) (Hagström, A. K. et al. A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory. Microb. Cell Fact. 12: 125 (2013)).

TABLE 7

Analytical parameters used for GC-MS analysis of DMDS-derivatives

System
Agilent 6890 N GC, ChemStation G1701EA E.02.01.1177

Column
Rtx-5 30 m × 320 μm × 25 μm

Pressure = 11.74 psi; Flow = 7.1 mL/min

Inlet
Heater = 250° C.; Pressure = 11.74 psi;

Total Flow {He} = 19.5 mL/min

Carrier
He @ 147 cm/sec, 11.74 psi

Signal
Data rate = 2 Hz/0.1 min

Oven
150° C. for 2 min

Ramp 10° C./min to 180° C.

Ramp 3° C./min to 260° C.

Ramp 20° C./min to 280° C., hold 10 min

Injection
Split, 250° C.

Split ratio −1:1

Detector
HP 5973 MSD in SCAN mode (mass range: 41 to 550 amu)

100 msec Dwell, EMV mode: Gain factor 1,

3 min solvent delay, 8.33 cycles/sec

Sample
Injection volume = 1 uL

Example 5: S. cerevisiae as a Production Platform for Insect Fatty Alcohol Synthesis

Background and Rationale

Engineering microbial production of insect fatty alcohols from fatty acids requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects as well as some microalgae. A number of gene variants were screened to identify enzyme activities that allow the creation of pathways capable of high level synthesis of a single or a blend of insect fatty alcohols. Additionally, these enzymes were screened across multiple hosts (Saccharomyces cerevisiae, Candida tropicalis, and Yarrowia lipolytica) in order to find a suitable host for optimum expression of these transmembrane proteins.

Summary of Approach

S. cerevisiae was engineered previously to express select functional transmembrane desaturase variants to allow synthesis of (Z)-11-hexadecenoic acid from palmitic acid. This allowed the identification and rank-ordering of the variants based on their bioconversion performance (see Example 4).

S. cerevisiae was engineered previously to express select functional transmembrane reductase variants to allow synthesis of (Z)-11-hexadecenol (Z11-16OH) from (Z)-11-hexadecenoic acid. This allowed the identification and rank-ordering of the variants based on their bioconversion performance (see Example 3).

Several fatty alcohol pathways comprised of the most active variant desaturases and reductases identified in the previous screens were assembled.

S. cerevisiae W303A and ΔOLE1 were transformed with the pathway constructs. Functionality of the pathway was assessed via the ability of the recombinant yeasts to synthesize Z11-16OH from palmitic acid.

GC-MS analysis was used to identify and quantify metabolites.

Results

The goal was to engineer one or more insect fatty alcohol biosynthetic pathways in S. cerevisiae. Previously, the functional expression of several transmembrane desaturases of insect origin in S. cerevisiae was demonstrated (see Example 4). Briefly, heterologous desaturase expression was enabled by designing an expression cassette which consists of an OLE1 promoter region, an N-terminal leader sequence encoding the first 27 amino acids of S. cerevisiae OLE1, and a terminator region of VPS13. Screening for active desaturases was done by using two approaches. First, active desaturases were screened for their ability to rescue ΔOLE1 growth without exogenous addition of unsaturated fatty acid (UFA), and second, active desaturases were screened via an in vivo screen for bioconversion of palmitic acid into (Z)-11-hexadecenoic acid. These screening strategies allowed the identification of several active variants, and the rank ordering of their relative activity. Based on these screening results, desaturases from Trichoplusia ni (TN_desat) and S. cerevisiae (SC_desat) were selected for combinatorial expression in fatty alcohol pathways. S. cerevisiae desaturase is known to form palmitoleic acid and oleic acid.

The functional expression of several transmembrane alcohol forming reductases of insect origin in S. cerevisiae had also been previously demonstrated (see Example 3). An expression cassette comprising the GAL1 promoter and CYC terminator was used to enable the functional expression of the reductases in S. cerevisiae. Screening several reductases via in vivo bioconversion of (Z)-11-hexadecenoic acid into Z11-16OH allowed the identification of active variants and rank ordering of their relative activity. Based on this screen, reductases from Helicoverpa armigera (HA_reduc), and Spodoptera littoralis (SL_reduc) were chosen for assembly of the fatty alcohol pathways.

Combinatorial assembly created four fatty alcohol pathways, i.e. TN_desat-HA_reduc, TN_desat-SL_reduc, SC_desat-HA_reduc, and SC_desat-SL_reduc. Pathways with SC_desat served as negative control for insect Z11-16OH synthesis. S. cerevisiae ΔOLE1 and W303A were transformed with constructs harboring these pathways, and transformants that grew on CM-Ura with 2% glucose and coated with palmitoleic acid were isolated. To test for fatty alcohol production, individual clones were inoculated into CM-Ura medium containing 2% glucose, 1% raffinose, 2% galactose, 300 mg/L palmitic acid, and 360 mg/L palmitoleic acid were added as bioconversion substrates. Bioconversion using palmitic acid without palmitoleic was also tested. Post ˜96h-cultivation in the presence of palmitic and palmitoleic acid, culture broth analysis revealed synthesis of Z11-9OH as a major C₁₆alcohol product at ˜0.2 mg/L, and ˜0.3 mg/L in cultivation of ΔOLE1 strains harboring SC_desat-HA_reduc, and TN_desat-HA_reduc, respectively (FIG. 15, FIG. 16). A minute amount of Z11-16OH was also detected in pathways with T. ni or S. cerevisiae desaturase, and H. armigera reductase. In general, it was expected that in the presence of palmitic acid and palmitoleic acid, Z9-16OH synthesis was more favorable than Z11-16OH synthesis because (Z)-11-hexadecenoic acid must be biosynthesized from T. ni desaturase, whereas exogenous addition of palmitoleic acid resulted in a more readily available substrate for synthesis of Z9-16OH. Fatty acid analysis was also performed. The results showed higher accumulation of (Z)-11-hexadecenoic acid (FIG. 17) in pathways containing insect desaturase than in pathways expressing S. cerevisiae desaturase. Albeit at minute quantities, detection of Z11-16OH, and (Z)-11-hexadecenoic acid from pathways harboring S. cerevisiae desaturase (which was unexpected) opens the possibility of a minor A11 desaturation activity by S. cerevisiae desaturase. Low level synthesis of Z11-16COOH fatty acid moieties can also be derived from elongation of Z9-14COOH fatty acyl intermediate. The data shown in FIG. 15 also showed that in comparison to pathways with H. armigera reductase, the inclusion of S. littoralis reductase resulted in the reduction of (up-to ˜30 fold) in Z9-16OH titer. No Z11-16OH could be detected in pathways employing S. littoralis reductase. These results are consistent with the reductase screening assay, which showed superior bioconversion of (Z)-11-hexadecenoic acid using H. armigera reductase in comparison to S. littoralis reductase.

The bioconversion of palmitic acid was also tested alone (without exogenous addition of palmitoleic acid) by ΔOLE1 strains expressing TN_desat-HA_reduc and TN_desat-SL_reduc (FIG. 18). Culture broth analysis determined the synthesis of Z11-16OH as the dominant unsaturated C16 fatty acid product (FIG. 16). In this assay, up to 0.22 mg/L, and 0.05 mg/L Z11-16OH was synthesized by a pathway harboring H. armigera reductase and S. littoralis reductase, respectively. The biologically produced Z11-16OH also matched the retention time and exhibited the characteristic 297.3 m/z peak like the authentic standard Z11-16OH as determined by GC-MS (SIM). Therefore, the regio- and stereoisomer of the biologically produced Z11-16OH was confirmed (FIG. 19). Furthermore, Z9-16OH (0.01 mg/L) was also observed in the cultivation of strain co-expressing T. ni desaturase and H. armigera reductase. This suggested that T. ni desaturase may also possess A9 desaturation activity.

OLE1 deletion impairs growth. Therefore, pathway expression was also explored in W303A, a host with intact OLE allele. However, despite growth improvement, pathway expression in this host resulted in more than two-fold reduction of Z11-16OH titers. This result was likely due to the repression of OLE1 promoter (which drove heterologous desaturase expression) by endogenous unsaturated fatty acyl:CoAs, the products of OLE1. The S. cerevisiae OLE1 promoter has been previously characterized with structural regions found to be positively and negatively regulated by saturated and unsaturated fatty acid, respectively (Choi, J-Y. et al. Regulatory Elements That Control Transcription Activation and Unsaturated Fatty Acid-mediated Repression of the Saccharomyces cerevisiae OLE1 Gene. J. Biol. Chem. 271: 3581-3589 (1996)). In addition to cis-transcriptional regulation, unsaturated fatty acids also interact with OLE1 promoter elements to regulate mRNA stability (Gonzales, C. I. et al. Fatty acid-responsive control of mRNA stability. Unsaturated fatty acid-induced degradation of the Saccharomyces OLE transcript. J. Biol. Chem. 271: 25801-25809 (1996)). Due to this inherent complexity of the OLE1 promoter, the utilization of unregulated orthogonal promoters, such as the OLE1 promoter from S. kluyveri (Kajiwara, S. Molecular cloning and characterization of the v9 fatty acid desaturase gene and its promoter region from Saccharomyces kluyveri. FEMS Yeast. Res. 2: 333-339 (2002)) to drive insect desaturase expression can be explored to enhance fatty alcohol production.

In summary, functional expression of synthetic pheromone pathway variants in S. cerevisiae ΔOLE1 resulted in the synthesis of Z11-16OH and Z9-16OH from palm oil fatty acids (palmitic acid and palmitoleic acid) up to approximately 0.2 mg/L and 0.3 mg/L, respectively.

The engineered pathway that resulted in the highest fatty alcohols is comprised of T. ni desaturase and H. armigera reductase.

Accumulation of (Z)-1-hexadecenoic acid, an intermediate of the pathway, was also observed in strains that produced Z11-16OH.

No Z11-16OH was produced and only trace Z9-16OH was detected in the negative control strain (harboring vector only).

The regio- and stereochemistry of the biologically produced Z11-16OH were confirmed by comparing the retention time and fragmentation pattern to the authentic standard compound via GC-MS.

Conclusions

The engineering of Baker's yeast for synthesis of Z11-16OH and Z9-16OH, fatty alcohol precursors of insect pheromones, was demonstrated.

Fatty alcohol production varies depending on the selection of the desaturase and reductase variants.

Accumulation of (Z)-11-hexadecenoic acid suggested the possibility of further fatty alcohol improvement by increasing the performance of alcohol forming reductase. However, it is also possible that detection of (Z)-11 l-hexadecenoic acid was due to its incorporation as phospholipid into any membrane other than the endoplasmic reticulum membrane (such as mitochondrial membranes, peroxisome, nuclear envelope, etc), therefore inaccessible to alcohol forming reductase (presumably translocated into the endoplasmic reticulum) which must utilize (Z)-11-hexadecenoic acid in its CoA thioester moiety as its substrate.

Culture conditions can be explored to increase fatty alcohol titers. The T. ni desaturase can be replaced in the pathway by A. transitella desaturase, another variant that also showed high activity and rescued ΔOLE1 growth faster than T. ni desaturase. The synthetic pathway can be imported into Candida tropicalis and Yarrowia lipolytica, which are yeasts with high adhesion property to hydrophobic substrates such as palmitic and palmitoleic acid. By increasing substrate accessibility to the microbial production platform, it is foreseeable that product titer and yield can be improved.

Materials & Methods

Strain Construction and Functional Expression Assay

S. cerevisiae ΔOLE1 (MATA OLE1::LEU2 ura3-52 his4), and W303A (MATA ura3-1 trp1-1 leu2-3_112 his3-11_15 ade2-1 can1-100) were used as expression hosts. Modular design allows combinatorial pathway assembly utilizing BamHI and XhoI to excise reductase synthons (see Example 3) and subcloning into plasmids containing pOLE1-desaturase constructs (see Example 4). Competent yeasts were transformed with pathway constructs and plated on CM-Ura glucose agar plate (Teknova). In the case of ΔOLE1 transformation, colony plating utilized 20 mM CM-Ura glucose agar plates that were coated with 100 μL CM-Ura glucose medium containing 1% tergitol and 3 μL palmitoleic acid.

To assess functional expression, transformants were inoculated in ˜20 mL CM-Ura liquid medium containing 6.7 g/L of YNB, 2% glucose, 1% raffinose, and 2% galactose. Fatty acid substrates, i.e. palmitic acid (in ethanol), was added at a final concentration of 300 mg/L. Palmitoleic acid was added at a final concentration of 360 mg/L. Bioconversion assay proceeded for 96 h at 28° C. prior to GC-MS analysis.

Metabolite Extraction and GC-MS Detection

Fatty acid analysis was as described in Example 4, except that instead of extracting the sample two times, the sample was only extracted once with chloroform containing 1 mM methyl heptadecanoate (C17:0Me). Fatty alcohol analysis was as described in Example 3, except that instead of hexane (containing tetradecanedioic acid), chloroform (containing 1 mM methyl heptadecanoate) was used. The extraction time was reduced from 1 h to 20 s. Afterwards the samples were collected in a 1.8 mL GC vial and not in a 1.5 mL plastic tube. The mass spectrometer was used in SIM mode (m/z 208, 297.3 and 387.3).

Example 6: Expression of Transmembrane Desaturases in Candida tropicalis

Background and Rationale

Engineering microbial production of insect fatty alcohols from fatty acids requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects as well as some microalgae. A number of gene variants were screened to identify enzyme activities that allow the creation of pathways capable of high level synthesis of a single or a blend of insect fatty alcohols. Additionally, these enzymes can be screened across multiple hosts (Saccharomyces cerevisiae, Candida tropicalis, and Yarrowia lipolytica) to optimize the search toward finding a suitable host for optimum expression of these transmembrane proteins.

Summary of Approach

A small set of desaturases (insect origin: Agrotis segetum, Amyelois transitella, Helicoverpa zea, Trichoplusia ni, Ostrinia furnacalis, and Lampronia capitella and marine diatom: Thalassiosira pseudonana) were selected as a test case to explore and establish functional expression assays, metabolite extraction methods, and analytical chemistry.

Successful integration and functional expression of mCherry control from pXICL expression cassette in SPV053 were confirmed.

A recombinant desaturase library using the same pXICL vector in SPV053 background was integrated (FIG. 20). One variant, the Z11 desaturase of Agrotis segetum, was also cloned to produce a protein product with the first 27 amino acids of Candida albicans Ole1p fused to the N-terminus of the insect desaturase (SEQ ID NO: 15).

Functionality of the desaturase was validated via an in vivo bioconversion of hexadecanoic acid (palmitic acid) into (Z)-11-hexadecenoic acid (palmitvaccenic acid).

GC-FID and GC-MS analyses were used to identify and quantify metabolites.

Results

Library Construction

This study focused on the screening for transmembrane desaturase variants in C. tropicalis (SPV053). Five insect desaturases with reported Z11 desaturase activity on palmitoyl-CoA (C16:0) (SEQ ID NOs: 16-19, 23) and three insect desaturases with reported Z9 desaturase activity (SEQ ID NOs: 20-22) were included in the screen. One variant, the Z11 desaturase from A. segetum (SEQ ID NO: 16), was also cloned with 27 amino acids of the Candida albicans OLE1 N-terminus fused upstream of the insect sequence (FIG. 20, SEQ ID NO: 15). At the time of construction, the A. segetum Z11 desaturase was believed to be a positive control and the C. albicans OLE1 fusion was constructed to test if inclusion of a Candida leader sequence would improve functional expression. The construct was designed to mimic those used in Saccharomyces cerevisiae desaturase screening (See Example 4). Finally, a control construct expressing mCherry red fluorescent protein (SEQ ID NO: 14) was included to act as a positive control for integration and expression and a negative control for recombinant desaturase activity (FIG. 21A-FIG. 21D).

Transformation efficiencies of linearized plasmids into SPV053 varied greatly across constructs. Despite low efficiencies, at least 3 clonal isolates were identified for each variant (Tables 8 and 9). It had been hypothesized that larger colonies on transformation plates were more likely to be positive integrants because the presence of the Zeocin resistance marker should increase growth rate under Zeocin selection. Analysis of the screening results suggested that the number of large colonies is not correlated to transformation efficiency. Instead total colony (small and large) count correlated best with observed efficiency (FIG. 22). In addition, in some cases positive clones were found among the small colonies. It is possible that at lower plating density growth rate may be correlated with integration events (i.e. positive integrants grow faster). A secondary screen of repatching colonies on YPD+Zeocin proved effective in enriching for positive integrants. Fast growing patches were more likely to be positive integrants than the general population of colonies on transformation plates.

TABLE 8

Desaturase transformations in SPV053. Efficiency of

transformation varied across constructs with a relatively

high degree of background under Zeocin selection.

large
total

colonies
colonies

pXICL
DNA
(control
(control

specificity
source species
plasmid
ug
plate)
plate)

control
mCherry_Ct
pPV0137
1.1
60 (60)
2,000 (600)

Z11

Agrotis segetum-
pPV0138
1.2
120 (78)
>10,000

OEL1_Ca

(320)

Agrotis segetum

pPV0139
1.3
115 (78)
8,000 (320)

Amyelois transitella

pPV0140
1.1
220 (78)
5,000 (320)

Trichoplusia nl

pPV0141
1.1
100 (78)
>10,000

(320)

Helicoverpa zea

pPV0142
1.0
350 (78)
5,000 (320)

Thalassiorira

pPV0146
1.1
140 (78)
1,500 (320)

pseudonana

Z9

Ostrinia furnacalis

pPV0143
0.9
220 (78)
6,000 (320)

Lampronia capitella

pPV0144
1.2
230 (78)
5,000 (320)

Helicoverpa zea

pPV0145
1.2
72 (78)
2,000 (320)

TABLE 9

Desaturase SPV053 library construction. Five insect

desaturases with putative Z11 desaturation activity and 3

insect desaturases with putative Z9 desaturation activity were

integrated into the SPV053 background using the

pXICL vector. In addition, a control strain

expressing mCherry was constructed with the same vector.

pXICL
Total
Total
Fraction

specificity
source species
plasmid
positives
screened
positive

control
mCherry_Ct
pPV0137
16
16
1.00

Z11

Agrotis segetum-
pPV0138
7
12
0.58

OEL1_Ca

Agrotis segetum

pPV0139
12
12
1.00

Amyelois transitella

pPV0140
5
60
0.08

Trichoplusia nl

pPV0141
6
12
0.50

Helicoverpa zea

pPV0142
5
120
0.04

Thalassiorira

pPV0146
3
96
0.03

pseudonana

Z9

Ostrinia furnacalis

pPV0143
3
57
0.05

Lampronia capitella

pPV0144
3
58
0.05

Helicoverpa zea

pPV0145
3
94
0.03

Functional Expression Assay

Functional expression of the heterologous desaturases was characterized by a series of in vivo bioconversion experiments. C. tropicalis SPV053 derived stains expressing insect desaturases were cultured in rich (YPD) or defined (CM glucose) media supplemented with ethanol (for induction) and saturated acid substrates (palmitic acid, methyl palmitate, methyl myristate). Small scale (2 ml) cultures were cultivated for a total of 72 hours in 24 deep well plates with substrate added after the initial 24 hours.

The first screen examined multiple bioconversion media with supplementation of a palmitic acid substrate. Two functional palmitoyl-CoA (Z)-11 desaturases were identified by fatty acid methyl ester (FAME) analysis of the cellular lipid content. Strains expressing A. transitella or H. zea Z1 desaturases (SPV0305-SPV0310) produced a fatty acid species not observed in the mCherry control strains (SPV0302-SPV0304) which eluted with the (Z)-11-hexadecenoic acid standard (FIG. 23). No other tested strains produced non-native fatty acid species (data not shown). Approximate fatty acid composition of the C16-fraction is listed in Table 10. The native palmitoyl-CoA (Z)-9 desaturase is still present in the SPV053 background which means the (Z)-9/(Z)-11 specificity of the desaturases cannot be rigorously determined. Supplementation of palmitic acid in the media increased the (Z)-11/(Z)-9 hexadecenoic acid ratio from 0.6 to 1.4 for H. zea desaturase expressing strains. (Z)-11-hexadecenoic acid titers were observed to be approximately 5.62 mg/L for strains expressing A. transitella desaturase and 5.96 mg/L for strains expressing H. zea desaturase. Similar performance was observed with methyl palmitate supplementation (data not shown).

TABLE 10

Composition of the C16-fatty acid fraction in different C. tropicalis

SPV053 expressing different desaturases. *NS = no substrate

(hexadecanoic acid) was added.

C16:0
Z9-C16:1
Z11-C16:1
Z11/Z9

[%]
[%]
[%]
ratio

mCherry
72.9
27.1
0.0
0.0

Hzea-YPD_NS
50.0
30.9
19.1
0.6

Hzea-YPD
58.1
17.5
24.4
1.4

AT-YPD
55.5
14.5
30.0
2.1

The bioconversion assay was scaled-up to 20 ml in shake flasks in order to generate enough biomass for additional characterization of the putative (Z)-11-hexadecenoic acid species. While the observed species eluted with the (Z)-11-hexadecenoic acid standard and independently of the (Z)-9-hexadecenoic acid standard, it was possible that a different fatty acid isomer (e.g. (E)-9-hexadecenoic acid) could have a similar retention time to (Z)-1-hexadecenoic acid. As different stereoisomers elute differently on the DB-23 the occurrence of (E)-11-hexadecenoic could be excluded. Final confirmation of (Z)-11-hexadecenoic acid production was completed by using mass spectroscopy detection of DMDS derivatized fatty acids to confirm the 11-regioselectivity. Using this derivatization technique (Z)-11 and (E)-11 isomers could in principle also be resolved. The fragmentation pattern of experimental samples could be matched to the (Z)-11-hexadecenoic acid standard (FIG. 24A-24E). Using this technique, production of the specific (Z)-11-hexadecenoic acid regio- and stereoisomer was confirmed for both A. transitella and H. zea desaturase expressing strains.

Finally, methyl myristate (C14:0) was tested as substrate for the entire desaturase library. A non-native fatty acid species which elutes between myristate (C14:0) and (Z)-9-tetradecenoic acid (Z9-C14:1) was observed in strains expressing either A. transitella or H. zea Z1 desaturases (FIG. 25A). It is hypothesized that this non-native species is (Z)-11-tetradecenoic acid, and this can be confirmed with an authentic standard. In addition, A. segetum Z11 desaturase, T. furnacalis Z9 desaturase, and H. zea Z9 desaturase all produced a shoulder peak which eluted just after the myristate (C14:0) peak (FIG. 25B). Other C14 derived species (e.g. tetradecanedioic acid) were observed in all strains. These results suggest that A. transitella and H. zea desaturases have some activity on myristoyl-CoA. Confirmation of unknown species and quantification is required to draw further conclusions about desaturase substrate specificity in vivo.

In summary, two desaturases from Helicoverpa zea (AAF81787) and from Amyelois transitella (JX964774), were expressed in SPV053 and conferred synthesis of (Z)-1-hexadecenoic acid from either endogenously produced or supplemented palmitic acid.

Functional expression of H. zea and A. transitella desaturases in C. tropicalis SPV053 was confirmed using an in vivo bioconversion assay in both rich (YPD) and defined (CM glucose) media. The active desaturases generated intracellular (Z)-11-hexadecenoic acid which was not observed in mCherry expressing control strains. C16-fatty acid composition of SPV053 expressing H. zea desaturase is approximately 50.0% hexadecanoic acid, 30.91% (Z)-9-hexadecenoic acid and 19.1% (Z)-11-hexadeceneoic acid. With palmitic acid supplementation the composition is 58.1% hexadecanoic acid, 17.5% (Z)-9-hexadecenoic acid and 24.4% (Z)-11-hexadeceneoic acid. The C16-fatty acid composition of SPV053 expressing A. transitella desaturase is 55.5% hexadecanoic acid, 14.5% (Z)-9-hexadecenoic acid and 30.0% (Z)-11-hexadeceneoic acid. In comparison, SPV053 expressing mCherry produced a C16-fatty acid composition of approximately 72.9% hexadecanoic acid, 27.1% (Z)-9-hexadecenoic acid and no (Z)-11-hexadeceneoic acid. (Z)-11-hexadecenoic acid was produced at approximately 5.5 mg/L in both strains expressing functional Z11 desaturases.

No (Z)-11-hexadecenoic acid was observed in strains harboring T. ni. T. pseudonana, or A. segetum desaturase.

No difference in fatty acid composition was observed for strains expressing Z9 insect desaturases from H. zea. O. furnacalis, or L. capitella.

The regio- and stereoisomer of the biologically produced (Z)-11-hexadecenoic acid were confirmed by comparing the retention time and fragmentation pattern of the authentic standard compound via GC-MS after DMDS derivatization.

Bioconversions of SPV053 expressing A. transitella and H. zea desaturases with supplementation of methyl myristate produced an unidentified metabolite not observed in the mCherry expressing negative control strain. The GC retention time of this metabolite is found between myristate (C14:0) and (Z)-9-tetradecenoic acid.

Conclusions

Functional expression of transmembrane desaturase of insect origin in C. tropicalis SPV053 has been achieved.

The active desaturases identified via screening in C. tropicalis also complemented OLE function when expressed in S. cerevisiae ΔOLE1 (See Example 4).

An in vivo assay can be used to assay desaturase activity in C. tropicalis for non-native fatty acid isomers (e.g. (Z)-11-hexadecenoic acid). Enhanced ratios of non-native fatty acids can be produced with supplementation of saturated acid substrates such as palmitic acid or methyl myristate.

Functional expression and/or activity of insect desaturases varies widely in C. tropicalis SPV053 depending on sequence origin. Similar to results observed in the S. cerevisiae screen (See Example 4), A. segetum and T. pseudonana variants did not produce detectable (Z)-11-hexadecenoic acid. Interestingly, T. ni desaturase also failed to produce detectable (Z)-11-hexadecenoic acid under assay conditions. Unlike in the S. cerevisiae assay, the T. ni expression construct did not include a chimeric OLE1 leader sequence.

The inclusion of the C. albicans OLE1 leader sequence on the functional H. zea variant and non-functional T. ni variant can be tested.

The functional expression of additional desaturase variants to identify C14-specific desaturases can be explored.

Expression of functional desaturase with reductase variants can be done and subsequent screen for unsaturated fatty alcohol production can be performed.

Materials & Methods

Strain Construction

A conservative approach was used for recoding of genes. Native sequences were unaltered except for replacement of CTG leucine codons with TTA. All genes were cloned into pPV0053 using NcoI and NotI restriction sites by Genscript. After transformation into E. coli NEB10β, plasmids were miniprepped using the Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, Calif.). Plasmids were linearized by digestion with BsiWI (New England Biolabs, Ipswich, Mass.) before transformation into SPV053. After digestion, DNA was isolated using Clean and Concentrator Kit (Zymo Research, Irvine. Calif.). Approximately 1 μg of DNA was transformed by electroporation. Instead of incubation with TE+100 mM lithium acetate+DTT, cells were incubated in only TE+100 mM lithium acetate for 2 hours. Positive integrants were found to be site-specific and genotyping was conducted by check PCR. A two-stage approach was adopted for further screening of low efficiency transformations. Approximately 60 colonies were re-patched on YPD+300 μg/ml Zeocin and grown overnight. The subset of patches which grew quickly (dense growth within 24 hours) were screened by colony PCR. The vast majority of rapid growing patches were identified as positive integrants.

Functional Expression Assay

Palmitic Acid Supplementation in YPD and CM Glucose

Positive isolates were re-patched onto YPD+300 pig/ml Zeocin and grown overnight and then stored at 4° C. Strains were inoculated from patch plates into 2 ml of YPD in 24 deep well plates (square well, pyramid bottom). Three positive clones were inoculated for each desaturase variant and the mCherry expressing control strain. Deep well plates were incubated at 30° C., 1000 rpm, and 80% humidity in the Infors HT Multitron Pro plate shaker for 24 hrs. After 24 hrs of incubation, cultures were split into equal 1 ml volumes to make two sets of identical plates. Both sets of plates were pelleted by centrifugation at 500×g. One set of plates was resuspended in 2 ml of YPD+0.3% (v/v) ethanol and the second set was resuspended in 2 ml of CM glucose+0.3% ethanol. Ethanol was added at this stage to induce recombinant enzyme expression from the ICL promoter. Cultures were incubated for another 24 hours under the same conditions before 300 mg/L palmitic acid was added to cultures from a 90 g/L stock solution in ethanol. The result was the addition of a fresh 0.3% ethanol in conjunction with the palmitic acid. A subset of strains was also cultured without palmitic acid addition. These cultures had 0.3% ethanol added instead. All cultures were incubated for an additional 24 hrs before a final addition of 0.3% ethanol. After another 24 hr period of incubation, 1.5 ml of each culture was harvested in 1.7 ml microcentrifuge tubes and pelleted. Supernatant was saved in fresh tubes and pellets were processed as described below. A subset of supernatant samples was also extracted to look for free acid in the extracellular medium.

Repeated Screening with Alternate Substrates

The mCherry control and confirmed positive variants were rescreened using both palmitic acid and methyl palmitate as substrates. The culturing was conducted as described above with equimolar (1.17 mM) amounts of substrate added from ethanol stock solutions (methyl palmitate 94 g/L stock, 313 mg/L final concentration). The same protocol was also repeated with the full panel of strains using an 84 g/L stock of methyl myristate (C14:0). The final concentration of substrate was again 1.17 mM.

Confirmation of (Z)-11-Hexadecenoic Acid Isomer

The in vivo bioconversion assay was scaled up for confirmation of (Z)-11-hexadecenoic acid synthesis, 2 ml YPD seed cultures of strains SPV0302, SPV0303, and SPV0304 (mCherry), SPV0304, SPV0305, and SPV0306 (A. transitella Z11 desaturase), and SPV0307, SPV0308, and SPV0309 (H. zea Z11 desaturase) were grown overnight at 30° C., 1000 rpm, 80% humidity in the Infors HT Multitron plate shaker, 200 μl of overnight culture from each of the three clonal isolates was pooled and inoculated into a single 125 ml baffled flask containing 20 ml YPD. The resulting three flasks were grown for 24 hrs at 30° C. and 250 rpm (Infors Flask shaker). Cultures were pelleted by centrifugation at 500×g and resuspended in 20 ml of YPD+0.3% (v/v) ethanol and returned to 125 ml baffled shake flasks. Cultures were incubated for an additional 24 hours before addition of 300 mg/L palmitic acid in a 90 g/L stock in ethanol (221 μl per flask). After 24 hours of incubation another 0.3% (v/v) ethanol (221 μl) was added to each flask for sustained induction. Flasks were incubated for an additional 24 hours before cells were harvested for FAME analysis and DMDS derivatization.

Metabolite Extraction and GC-MS Detection

Total lipid composition as well as the (Z)-11-hexadecenoic acid quantification was based on modified procedures by Moss et al. (1982) and Yousuf et al (2010). The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL screw-cap GC-vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM heptadecanoic were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase were transferred into a new 1.5 mL plastic tube. The aqueous phase was extracted a second time with 500 μL chloroform, this time without heptadecanoic acid. The combined organic phases were evaporated at 90° C. After cooling to room temperature, residual fatty acid methyl esters and free fatty acids were dissolved and derivatized in methanol containing 0.2 M TMSH (trimethylsulfonium hydroxide).

The regioselectivity of biologically produced (Z)-11-hexadecenoic acid was determined by comparing the fragmentation patterns of the dimethyl disulfide (DMDS) derivative with the DMDS derivative of an authentic standard. A yeast culture was split into 12 aliquots (to not change any parameters in the developed procedure). The cells were pelleted, which yielded 63 mg cells (ccw) on average (755 mg from 18 mL culture). The pellets were subjected to base methanolysis as described above. However, after acidification the samples were combined in a 50 mL falcon tube. The combined sample was extracted two times with 10 mL chloroform. The mixture was centrifuged 10 min at 3000 rpm to achieve a better phase separation. The combined organic phases were combined in a new 50 mL falcon and were washed consecutively with 10 mL brine and 10 mL water. The organic phase was dried with anhydrous sodium sulfate and concentrated in vacuo. The concentrated oil was dissolved in 1.5 mL chloroform and transferred to a 1.5 mL plastic tube. The chloroform was evaporated at 90° C. The remaining sample was the dissolved in 50 μL methyl tert-butyl ether (MTBE). The 50 μL were split into 1, 5, 10 and 20 μL and transferred into GC-vials without insert. To each vial 200 μL DMDS (dimethyl disulfide) and 50 μL MTBE (containing 60 mg/mL iodine) were added. After the mixture was heated 48 h at 50° C., excess iodine was removed by the addition of 100 μL saturated sodium thiosulfate solution, however, due to excessive formation of detergents from the Candida strain, the layer did not mix properly. The samples were therefore diluted in a 15 mL falcon tube to a final sample composition of 200 μL, 3.55 mL MTBE (containing iodine and analyte), 500 μL dichloromethane, 1.5 mL water and 1 mL ethanol. The organic phase was evaporated stepwise at 85° C. in a 1.8 mL glass vial. The samples were taken up in 500 μL dichloromethane and the sample was analyzed by GC-MS using the method of Hagström et al. (2013) as in Example 4.

Example 7: Expression of Transmembrane Desaturases in Yarrowia lipolytica

Background and Rationale

Engineering microbial production of insect fatty alcohols from fatty acids requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase, and an alcohol-forming reductase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA, and subsequently into fatty alcohols. A number of genes encoding these enzymes are found in some insects as well as some microalgae. Alternatively, regio- and stereospecific desaturases can be used to produce a microbial oil rich in fatty acid precursors. The microbial oil can then be derivatized and reduced to active ingredients. A number of gene variants were screened to identify enzyme activities that allow the creation of pathways capable of high level synthesis of a single or a blend of insect fatty acids and alcohols. Additionally, these enzymes were screened across multiple hosts (Saccharomyces cerevisiae, Candida viswanathii (tropicalis), and Yarrowia lipolytica) to optimize the search toward finding a suitable host for optimum expression of these transmembrane proteins.

Initial screening of desaturases in S. cerevisiae and C. viswanathii (tropicalis) identified three active Z11-C16:1 desaturase variants from Amyelois transitella, Helicoverpa zea, and Trichoplusia ni. The S. cerevisiae screening used coding sequences with an N-terminal leader sequence of the S. cerevisiae Ole1p Z9 desaturase fused to the full length insect Z11 desaturase sequence. This strategy has been used previously in the scientific literature to express eukaryotic desaturases in S. cerevisiae. All three of the above desaturases displayed Z11 desaturase activity with the Ole1p leader fusion when expressed in a OLE1 deletion background. An analogous design with a C. albicans Ole1p leader sequence was used with the Z11 desaturase from H. zea. While active, this Ole1p-H. zea desaturase fusion did not significantly increase Z11-hexadecenoic acid titer. Additionally, a conservatively optimized A. transitella Z11 desaturase was active in both S. cerevisiae and C. viswanathii. The following study focused on testing the functional expression of the H. zea, T. ni, and A. transitella Z11 desaturases in two different Y. lipolytica strains, SPV140 and SPV300. Both native and Homo sapiens codon optimized sequences were used for the H. zea and T. ni desaturases while only the native sequence was used for A. transitella. Finally, the N-terminus of the Y. lipolytica Ole1p Z9 stearoly-CoA desaturase aligns more closely with insect desaturases than the N-terminus of Ole1p from either S. cerevisiae or C. albicans. Based on this alignment two additional desaturase versions were created. A putative leader sequence was swapped from the Y. lipolytica Ole1p onto the T. ni and H. zea desaturases.

Summary of Approach

A focused library of Z11 desaturases (insect origin: Amyelois transitella. Helicoverpa zea. Trichoplusia ni), which had observed activity in either S. cerevisiae or C. vswanathii were cloned into a double crossover cassette targeting the XPR2 locus with a URA3 selection marker. Protein coding sequences use either the native insect sequence (SEQ ID NOs: 24, 25), Homo sapiens optimized coding sequence (SEQ ID NOs: 26, 27), or the Homo sapiens optimized sequence with the N-terminal 84 bases (H. zea, SEQ ID NO: 29) or 81 bases (T. ni. SEQ ID NO: 28) swapped for the N-terminal 96 bases of the Y. lipolytica OLE1 (YALI0C05951) gene. Unlike in the S. cerevisiae and C. viswanathii screens, the leader sequence chimeras test a direct swap of leader sequences instead of concatenating a host leader sequence to the N-terminus of the full length desaturase coding sequence. Only the native coding sequence was used for the A. transitella desaturase (SEQ ID NO: 30).

Each of the 7 desaturase constructs was transformed into SPV140 (PO1f) and SPV300 (H222 ΔP ΔA ΔF ΔURA3) and site-specific integrants were confirmed.

Desaturase activity was tested via an in vivo bioconversion of hexadecanoic acid (palmitic acid) into (Z)-11-hexadecenoic acid (palmitvaccenic acid) in YPD medium.

GC-FID Analyses were Used to Identify and Quantify Metabolites.

Results

Strain Construction

Desaturase variants were cloned into the pPV101 vector which contains a Y. lipolytica expression cassette targeting integration into the XPR2 locus (YALI0F31889g).

The T: ni and H. zea desaturases were each synthesized with the native insect sequence (SEQ ID NOs: 24, 25), full length insect sequence codon optimized for Homo sapiens (SEQ ID NOs: 26, 27), or with the putative leader sequence replaced by the leader sequence from Y. lipolytica OLE desaturase (SEQ ID NOs: 28, 29). The A. transitella desaturase was also synthesized using the native insect coding sequence (SEQ ID NO: 30). All seven desaturase variants were transformed into SPV140. Based on previous activity results, only the H. zea and A. transitella desaturase variants were transformed into SPV300.

Functional Expression Assay

Functional activity was assessed by a modification of the protocol used for transmembrane desaturase expression in C. viswanathii SPV053 (See Example 6). Briefly, Y. lipolytica SPV140 and SPV300 derived stains expressing insect desaturases were cultured in rich (YPD) to generate biomass. Using the YPD generated biomass, small scale (2 ml) cultures were cultivated with palmitic acid for a total of 48 hours in 24 deep well plates (See Materials & Methods for detail).

In the initial screen of T. ni, H. zea, and A. transitella variants, only H. zea desaturase variants that were codon optimized for Homo sapiens produced detectable Z11-hexadecenoic acid (FIG. 26). Expression of native H. zea desaturase conferred production of 100+5 mg/L Z11-hexadecenoic acid and the version with a Y. lipolytica OLE leader sequence produced 83±11 mg/L. As seen in FIG. 26, the distribution of the other major fatty acid species was relatively unaffected by functional desaturase expression. In the active strains, Z11-hexadecenoic acid made up ˜10% (g/g) of the fatty acid species (including palmitic acid substrate which may be adsorbed to the outer cell surface).

A follow up experiment was conducted comparing active variants in the SPV140 background to SPV300 derived desaturase strains. The parent SPV300 and SPV140 expressing hrGFP were used as negative controls. The same bioconversion assay protocol was used. As in SPV140, only H. sapiens optimized variants produced detectable activity (FIG. 27). SPV300 strains grew to higher final cell densities (SPV300 OD600=26-28, SPV140 OD600=19-22) (FIG. 28). The highest titers were observed for strains expressing the native H. zea Z11-desaturase with H. sapiens codon optimization (pPV199). The retested SPV140 strains produced 113±1 mg/L (5.5±0.2 mg/L/OD) Z11-hexadecenoic acid which is 13% higher than titers observed in the first experiment (FIG. 29). SPV300 strains expressing the same desaturase generated a wider range of productivity. On average they produced 89±18 mg/L (3.3±1.2 mg/L/OD) Z11-hexadecenoic acid, but one clone produced 124 mg/L (4.6 mg/L/OD) Z11-hexadecenoic acid.

In summary, only the H. zea Z11 desaturase variants with Homo sapiens codon optimization produced detectable Z11-hexadecenoic acid. Under the current assay condition, marginally higher titers were observed in the SPV140 background over SPV300. Table 11 summarizes the Z11-hexadecenoic acid titers.

TABLE 11

Z11-hexadecenoic acid titers obtained from expression of exemplary

desaturases in Yarrowia lipolytica

Codon
Parent
Z11-hexadecenoic

Desaturase
optimization
Strain
acid titer (mg/L)

Z11 T. ni
Native
SPV140
ND (no detection)

Z11 T. ni

Homo sapiens

SPV140
ND

YI OLE1-Z11 T. ni

Homo sapiens

SPV140
ND

Z11 H. zea
Native
SPV140
ND

SPV300
ND

Z11 H. zea

Homo sapiens

SPV140
100 ± 5

SPV300
87 ± 18

YI OLE1-Z11 H. zea

Homo sapiens

SPV140
83 ± 11

SPV300
55 ± 1

Z11 A. transitella
Native
SPV140
ND

SPV300
ND

In SPV300, one non-site-specific integrant of pPV200 (Y. lipolytica OLE1-H. zea Z1 desaturase with Homo sapiens codon optimization) was tested. This integrant did not produce detectable Z11-hexadecenoic acid, while the two site-specific integrants produced 55+1 mg/L.

No major hydroxy or diacid peaks were observed from pellets of SPV140 or SPV300 derived strains, and deletion of β-oxidation/ω-oxidation genes in SPV300 did not increase Z11-hexadecenoic acid accumulation under the current assay condition (relatively low substrate concentration, rich medium).

Conclusions

The H. zea Z11 desaturase is active and confers production of ˜100 mg/L Z11-hexadecenoic acid, from ˜500 mg/L palmitic acid substrate. The functional expression was demonstrated across three positive integrants and replicate experiments in a 24 well plate assay.

H. zea desaturase required codon optimization (Homo sapiens or potentially Y. lipolytica) for activity in Y. lipolytica.

The T. ni Z11 desaturase, while active in S. cerevisiae, does not produce detectable Z11-hexadecenoic acid in Y. lipolytica.

The reproducibility of the assay for Y. lipolytica strains can be confirmed starting from glycerol stock.

A. transitella desaturase can be codon optimized for expression in Y. lipolytica.

Since Y. lipolytica is a candidate production host, additional copies of active desaturases can be integrated in Y. lipolytica, culture conditions to improve bioconversion can be identified, and substrate conversion can be quantified.

Materials & Methods

Strain Construction

All desaturase genes were synthesized (Genscript). Either native sequences or Homo sapiens codon optimization was used. Synthesized genes were subcloned into pPV101. Plasmids were transformed and prepped from E. coli EPI400 using the Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, Calif.). Approximately ˜1-2 μg of linearized DNA was transformed using Frozen-EZ Yeast Transformation II Kit (Zymo Research. Irvine, Calif.). The entire transformation mixture was plated on CM glucose-ura agar plates. Positive integrants were found to be site-specific and genotyping was conducted by check PCR.

Functional Expression Assay

Palmitic Acid Supplementation in YPD

Positive isolates were re-patched onto YPD, grown overnight, and then stored at 4° C. Strains were inoculated from patch plates into 2 ml of YPD in 24 deep well plates (square well, pyramid bottom). Three positive clones were inoculated for each desaturase variant. Three isolates of pPV101 in SPV140 and the parent SPV300 were used as negative controls. Deep well plates were incubated at 28° C. and 250 rpm in the Infors Multitron refrigerated flask shaker for 24 hrs. After 24 hrs of incubation, a 1 ml volume of each culture was pelleted by centrifugation at 500×g. Each pellet was resuspended in 2 ml of YPD, 500 mg/L palmitic acid was added to cultures from a 90 g/L stock solution in ethanol. The result was the addition of 0.5% ethanol with the palmitic acid substrate. All cultures were incubated for 48 hours before endpoint sampling. Final cell densities were measured with the Tecan Infinite 200pro plate reader, 0.75 or 0.8 ml of each culture was harvested in 1.7 ml microcentrifuge tubes and pelleted. Supernatant was removed and pellets were processed as described below.

Metabolite Extraction and GC-FID Analysis

Total lipid composition as well as the (Z)-11-hexadecenoic acid quantification was based on modified procedures by Moss et al. (1982) and Yousuf et al (2010). The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL crimp vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM methyl heptadecanoate were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase were transferred into a GC vial. For the analysis of lipids and the quantification of fatty acids 50 L of 0.2 M TMSH (trimethylsulfonium hydroxide in methanol) was added and the sample analyzed by GC-FID.

Example 8: Candida viswanathii (Tropicalis) as a Production Platform for Insect Fatty Alcohol Synthesis

Background and Rationale

Variants of insect transmembrane desaturases and reductases were previously screened and rank-ordered based on their functional expression in either Candida viswanathii or Saccharomyces cerevisiae (see Examples 3, 4 and 6). Helicoverpa zea desaturase and Helicoverpa armigera reductase were selected to assemble a synthetic insect fatty alcohol pathway in C. viswanathii. Simultaneous expression of codon optimized H. zea desaturase under Candida isocitrate lyase (ICL) promoter, and codon optimized H. armigera reductase under Candida transcription elongation factor (TEF) promoter was achieved via genomic integration of the full fatty alcohol pathway. Accumulation of Z11-16OH was achieved in cultures of the recombinant strain (SPV0490) using simple carbon sources and palmitic acid.

Summary of Approach

Integration plasmids were designed containing a functional Helicoverpa zea desaturase (See Example 6) paired with a Helicoverpa armigera reductase driven by a putatively constitutive C. tropicalis promoter (pTEF).

Functionality of the full pathway was assessed via an in vivo bioconversion of hexadecanoic acid (palmitic acid) into Z11-16OH.

GC-FID and GC-MS analyses were used to identify and quantify metabolites.

Results

Accumulation of Z11-16OH was detected in cultures of Candida engineered to express H. zea desaturase under an ICL promoter and H. armigera reductase under a TEF promoter (Table 12 and FIG. 30).

TABLE 12

Tabulated Z11-16OH titers from Candida viswanathii

bioconversion assay. SPV088 is C. viswanathii which was engineered

to express mCherry (negative control).

SPV0490 is C. viswanathii which was engineered

to express the insect fatty alcohol pathway.

Z11-16OH titers (mg/L)

SPV0488

negative control)
SPV0490

Sample 1
0.08
1.03

Sample 2
0.07
0.93

Sample 3
0.06
0.88

Average
0.07
0.95

StDev
0.01
0.06

Materials & Methods

Strain Construction

The integration plasmid (ppV0228) was designed to contain two expression cassettes. The first cassette contains H. zea codon-optimized desaturase (SEQ ID NO: 31) that was driven by the C. viswanathii ICL promoter (SEQ ID NO: 33). The second cassette contains codon-optimized H. armigera reductase (SEQ ID NO: 32) driven by the C. tropicalis TEF promoter (SEQ ID NO: 34). Gene expression in the ICL promoter cassette is terminated by the ICL terminator sequence (SEQ ID NO: 35). Gene expression in the TEF promoter cassette is terminated by the TEF terminator sequence (SEQ ID NO: 36). A conservative approach was used for recoding of genes. Native gene sequences were unaltered except for replacement of CTG leucine codons with TTA. After transformation into E. coli NEB10β, plasmids were miniprepped using the Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, Calif.). Plasmids were linearized by digestion with BsiWI (New England Biolabs, Ipswich, Mass.) before transformation into SPV053. After digestion, DNA was isolated using Clean and Concentrator Kit (Zymo Research, Irvine, Calif.). Approximately 3-5 μg of DNA was transformed by electroporation. Positive integrants were found to be site-specific and genotyping was conducted by check PCR. A two-stage approach was adopted for further screening of low efficiency transformations. Approximately 100 colonies were re-patched on YPD+250 μg/ml Zeocin and grown overnight. The subset of patches which grew quickly (dense growth within 24 hours) were screened by colony PCR.

Functional Expression Assay

Palmitic Acid Supplementation in YPD

Positive isolates were re-patched onto YPD+300 μg/ml Zeocin, grown overnight and then stored at 4° C. Strains were inoculated from patch plates into 2 ml of YPD in 24 deep well plates (square well, pyramid bottom). Four positive clones were inoculated for each desaturase and reductase variant and three positive clones were inoculated for each desaturase and mCherry expressing control strain. Deep well plates were incubated at 30° C., 1000 rpm, and 80% humidity in the Infors HT Multitron Pro plate shaker for 24 hrs. After 24 hrs of incubation, a 1 ml volume of each culture was pelleted by centrifugation at 500×g. Each pellet was resuspended in 2 ml of YPD+0.3% (v/v) ethanol. Ethanol was added at this stage to induce recombinant enzyme expression from the ICL promoter. Cultures were incubated for another 24 hours under the same conditions before 300 mg/L palmitic acid was added to cultures from a 90 g/L stock solution in ethanol. The result was the addition of a fresh 0.3% ethanol in conjunction with the palmitic acid. All cultures were incubated for an additional 24 hrs before a final addition of 0.3% ethanol. After another 24 hr period of incubation, 1.5 ml of each culture was harvested in 1.7 ml microcentrifuge tubes and pelleted. Supernatant was removed and pellets were processed as described below.

Metabolite Extraction and GC-MS Detection

The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL crimp vial. The mixture was heated for 1 h in a heat block at 90° C. Prior to acidification with 400 μL 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM methyl heptadecanoate were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase were transferred into a GC vial. The organic phase was evaporated in a heat block at 90° C. for 30 min. The residue was dissolved in 50 μL N,O-Bis(trimethylsilyl)trifluoroacetamide containing 10/trimethylchlorosilane. Prior to transfer into glass inserts the mixture was heated 5 min at 90° C. The samples were analyzed by GC-MS (Table 13).

TABLE 13

Analytical parameters used for GC-MS analysis of metabolites

System
Agilent 6890 N GC, ChemStation G1701EA E.02.01.1177

Column
DB23 30 m × 25 μm × 25 μm

Pressure = 11.60 psi; Flow = 0.6 mL/min

Inlet
Heater = 250° C.; Pressure = 11.74 psi;

Total Flow {He} = 111 mL/min

Carrier
He @ 29 cm/sec, 11.60 psi

Signal
Data rate = 2 Hz/0.1 min

Oven
150° C. for 1 min

Ramp 12° C./min to 220° C., hold 3 min

Ramp 35° C./min to 300° C., hold 4 min

Injection
Splitless, 250° C.

Detector
HP 5973 MSD in SIM mode (m/z: 208.0, 297.3 and 387.3),

100 msec Dwell, EMV mode: Gain factor 1,

2.4 min solvent delay, 3.09 cycles/sec

Sample
Injection volume = 1 μL

Example 9: Insect Fatty Alcohol Production from Yarrowia lipolytica

Background and Rationale

Yarrowia lipolytica was engineered as a production platform for insect fatty alcohol (Z11-16OH and Z9-16OH) synthesis from palmitic acid.

After individually confirming functional expression of a Z11 desaturase (Example 7) and fatty acyl-CoA reductase (FAR), the full Z11-16OH and Z9-16OH pathways (Bdr) were engineered in Y. lipolytica. For the purpose of improving fatty alcohol titers, cultivations designed for promoting growth vs. for eliciting lipid storage were also explored. A growth condition favors high biomass production, but limits fatty acyl-CoA pool size used by the engineered pathway and directs fatty acyl-CoA intermediates to membrane synthesis. Conversely, a lipid storage condition creates a strong sink for production of fatty acyl-CoAs which is desirable. However, fatty acyl-CoA transport towards lipid bodies creates a strong competition for FAR activity. Under this second scenario, even though Z11-16Acid or Z9-16Acid accumulates in the cell, most of it is inaccessible to the FAR. On the other hand, there may be a continual flux of lipid remobilization under lipid storage conditions which leads to a sustained pool of Z11-16CoA or Z9-16CoA which is available to the FAR.

Summary of Approach

Two biodesaturation-reduction (Be) pathway variants were tested in the H222 ΔPΔAΔF (SPV300) background. The first combined recombinant expression of Helicoverpa zea Z11 desaturase paired with a Helicoverpa armigera fatty acyl-CoA reductase (FAR amino acid sequence set forth in SEQ ID NO: 41) creating a Z11-16OH synthesis pathway. The second combined native Y. lipolytica Z9 desaturase activity with H. armigera fatty acyl-CoA reductase (FAR) expression creating a Z9-16OH pathway.

Two integration plasmids were constructed to express the H. zea desaturase and the H. armigera FAR. The TEF promoter was used for desaturase expression and the EXP1 (export protein) or the TAL (transaldolase) promoter was used for reductase expression.

Successful integration of the Z11-16OH pathway cassette into the H222 ΔPΔAΔF (SPV300) background was confirmed by colony PCR.

Functionality of the full Z11-16OH pathway was assessed via an in vivo bioconversion of 16Acid (palmitic acid) into Z11-16OH (Z-11-hexadecenol).

Functionality of a full Z9-16OH pathway was assessed via an in vivo bioconversion of 16Acid (palmitic acid) using previously constructed SPV471 (H222 ΔPΔAΔF derived) which expresses the H. armigera FAR driven by the TEF promoter.

GC-MS analysis was used to identify and quantify Z9-16OH and Z11-16OH. GC-FID analysis was used to identify and quantify fatty acids.

Summary

Ten isolates expressing the H. zea desaturase (pTEF) and H. armigera reductase (pEXP1) were screened. The in vivo bioconversion assay confirmed Z11-16OH production from all isolates.

Relatively low, detectable Z11-16OH titers (0.26±0.09 mg/L) were observed in a YPD medium supplemented with 10 g/L methyl palmitate. The Z11-16Acid precursor was measured at 220±11 mg/L (across clones 2, 4, 9, 17, 23).

Higher Z11-16OH titers were observed in a semi-defined medium with C:N ratio of ˜80. Across all 10 isolates Z11-16OH was produced at 2.65±0.36 mg/L. The Z11-16Acid precursor titer was 900±30 mg/L. One isolate (SPV578) produced 3.68±0.31 mg/L Z11-16OH (Z11-16Acid 840±14 mg/L).

Nine isolates expressing the H. zea desaturase (pTEF) and H. armigera reductase (pTAL1) were screened. The in vivo bioconversion assay confirmed Z11-16OH production from all isolates.

One isolate (SPV603) produced 6.82±1.11 mg/L Z-16OH in a semi-defined medium (Z11-16Acid 1.36 g/L).

The previously tested reductase strain, SPV471 (H222 ΔPΔAΔF expressing H. armigera FAR), produced 4.30±2.33 mg/L Z9-16OH and 450±80 mg/L Z9-16Acid using a semi-defined medium (C:N ratio of ˜80).

TABLE 14

Summary table of Z11/Z9-16OH titers from B_drpathway

strains in in vivo bioconversion assay.

Z11-16OH
Z9-16OH

Strain
Medium
(mg/L)
(mg/L)

pTEF-H. zea Z11
Semi-Defined
3.99 ± 0.37
0.22 ± 0.03

desaturase
C:N = 80
(n = 4)
(n = 4)

pEXP-H. armigera FAR

Clone 17 (SPV578)

pTEF-H. zea Z11
Semi-Defined
6.82 ± 1.11
0.22 ± 0.01

desaturase
C:N = 80
(n = 2)
(n = 2)

pEXP-H. armigera FAR

Clone 9 (SPV603)

pOLE1-Y.lipolytica
Semi-Defined
0.22 ± 0.03
4.30 ± 2.23

OLE1 (native)
C:N = 80
(n = 2)
(n = 2)

pTEF-H. armigera FAR

(SPV471)

Results

Strain Construction

Evidence in the literature suggests both insect desaturases and FARs are localized in the membrane of the endoplasmic reticulum with active sites oriented towards the cytoplasm. Of the functional variants, the Z11 desaturase from H. zea and the FAR from H. armigera (FAR amino acid sequence set forth in SEQ ID NO: 41) were selected, one hypothesis being that using enzymes from the same genus (Helicoverpa) could better conserve protein-protein interactions that may occur in the ER membrane.

Two new constructs were ordered from Genscript and cloned into the previously assembled H. zea desaturase plasmid, pPV0199. Two FAR synthons with either the EXP1 or TAL1 promoter from Y. lipolytica were cloned into this expression cassette.

One dual expression plasmid (with EXP1 promoter) was transformed into the parent strain SPV300 (H222 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1 Δura3). Two different competent cell preparations of the same parent strain were transformed to study variability in strain performance resulting from competent cell preparation. Approximately 25% of URA+ clones were confirmed to be targeted integrants at the XPR2 locus (20% for preparation 1, 33% for preparation 2). Two clones from Comp. Cell Preparation 1 and all eight targeted clones from Comp. Cell Preparation 2 were selected for screening in the functional expression assay.

The second dual expression plasmid (with TAL1 promoter) was integrated into the same parent strain (SPV300). Twenty-three colonies were screened by check PCR and 11 were found to be targeted integrants (48%). Nine integrants were selected for screening in the functional expression assay.

The construct of SPV471 (H222 ΔPΔAΔF expressing H. armigera FAR) was described previously.

Z11-16OH Functional Expression Assay

An in vivo, 24-well plate assay was used to evaluate production of Z11-16OH. The assay was based on designs used for screening desaturase and reductase variants as well as conditions used to increase fatty acid accumulation. A rich medium (YPD) and a semi-defined medium were used with 10 g/L methyl palmitate supplemented as bioconversion substrate. The semi-defined medium had a C:N ratio of ˜80 and included 5 g/L glycerol and 60 g/L glucose (See Materials & Methods for further details).

The initial screen of strains harboring the H. zea desaturase driven by the TEF promoter and the H. armigera FAR (FAR amino acid sequence set forth in SEQ ID NO: 41) driven by the EXP1 promoter confirmed that the presence of FAR was required to produce Z11-16OH. No hexadecenol was observed from both the parent and desaturase-only control strains under any condition. Under both media conditions Z11-16OH and to a lesser extent Z9-16OH were detected from clones expressing the full desaturase-reductase pathway. When the conversion was completed in rich medium, 0.26±0.09 mg/L Z11-16OH and 0.06±0.01 mg/L Z9-16OH were produced (FIG. 32A). A10-fold increase in Z11-16OH titer and 3-fold increase in Z9-16OH titer was observed when the Semi-Defined medium was used (FIG. 32B). Across all pathway clones 2.65±0.29 mg/L Z11-16OH and 0.18±0.02 mg/L Z9-16OH were produced. The enrichment of Z11-16OH over Z9-16OH supports the potential for engineering a regiospecific Bdr pathway. Consistency between technical replicates varied across clones under the Semi-Defined medium condition. Titers for Clones 2, 4, 6, 9, and 17 were consistent with CVs <20. Clones 1, 7, and 23 have CVs >40%. The highest consistent Z11-16OH titer was observed for Clone 17, 3.68±31 mg/L (Table 15).

TABLE 15

Summary table of Z11/Z9-16OH tilers for pEXP1 clones. A

population of ten isolates expressing the H.zea desaturase driven

by pTEF and H.armigera reductase driven by pEXP1, from

two independent competent cell preparations, were assayed for

Z11-16OH and Z9-16OH production under two different media

conditions. Alcohol production across isolates and from select

clones are presented.

pTEF-Hz_

Z11-16OH
Z9-16OH

desat

fold
fold

pEXP-Ha_

increase
increase

FAR

Z11-16OH
Z9-16OH
(relative
(relative

Clone(s)
Medium
(mg/L)
(mg/L)
to YPD)
to YPD)

All clones
YPD
0.26 ± 0.09
0.06 ± 0.01
—
—

All clones
Semi-
2.65 ± 0.29
0.18 ± 0.02
10
3

Defined

Clone 2
YPD
0.18 ± 0.09
0.05 ± 0.03
—
—

(SPV574)

Clone 2
Semi-
2.08 ± 0.26
0.14 ± 0.04
12
3

(SPV574)
Defined

Clone 4
YPD
0.28 ± 0.01
0.11 ± 0.01
—
—

(SPV575)

Clone 4
Semi-
3.24 ± 0.28
0.21 ± 0.03
12
2

(SPV575)
Defined

Clone 9
YPD
1.03 ± 0.84
0.05 ± 0.01
—
—

(SPV576)

Clone 9
Semi-
1.56 ± 0.28
0.11 ± 0.02
1.5
2

(SPV576)
Defined

Clone 23
YPD
0.16 ± 0.14
0.05 ± 0.05
—
—

(SPV577)

Clone 23
Semi-
3.35 ± 1.85
0.26 ± 0.15
21
5

(SPV577)
Defined

Clone 17
YPD
0.19 ± 0.01
0.06 ± 0.01
—
—

(SPV578)

Clone 17
Semi-
3.68 ± 0.31
0.26 ± 0.02
14
4

(SPV578)
Defined

The lipid profiles of the full pathway clones were also quantified. For simplicity the 16 carbon fatty acid species are plotted for select clones in FIG. 33A-33B. In general, the full Bdr pathway clones accumulated less Z11-16Acid than the desaturase only control (0.25<0.5 g/L in YPD, 0.8-1.0<1.5 g/L in Semi-Defined). Lower Z11-16Acid titers in full Bdr pathway clones may result from reduced desaturase expression in the dual expression cassette or potentially from Z11-16Acid consumption by FAR and subsequent byproduct pathways. No trend in 16Acid titer was observed in YPD, while 16Acid titers were similar for desaturase only and full pathway strains in the Semi-Defined medium.

Strains using the second dual expression cassette (pTAL-Ha_FAR) were assayed under the same Semi-Defined medium condition used to evaluate the pEXP clones. Nine pTAL clones were assayed against SPV300 (parent), SPV575 (pEXP-Ha_FAR Clone 4), and SPV578 (pEXP-Ha_FAR Clone 17) controls. As expected, no alcohol products were observed from the negative control. Alcohol titers from pEXP positive control strains replicated results observed in the initial assay of pEXP clones (FIG. 34, Table 16). Excluding one outlier clone, Clone 9. Z11-16OH titer was equivalent from pTAL clones (4.19±0.16 mg/L) and pEXP clones (4.10±0.22 mg/L). Clone 9 produced Z11-16OH at 6.82±1.11 mg/L. As in the first assay with pEXP clones, low but detectable titers of Z9-16OH were observed (FIG. 34, Table 16).

TABLE 16

Summary table of Z11/Z9-16OH titers for pTAL1 clones. A population

of nine isolates expressing the H.zea desaturase under the TEF promoter

and H.armigera reductase under the TAL promoter were assayed for

Z11-16OH and Z9-16OH production under a Semi-Defined medium

condition. Clones were compared to positive controls expressing the H.

zea desaturase under the TEF promoter and H.armigera reductase under

the EXP promoter. Alcohol production across isolates and from select

clones are presented.

pTEF-Hz-

desat

pEXP-Ha-

FAR

Z11-16OH
Z9-16OH

Clone(s)
Medium
(mg/L)
(mg/L)

EXP Clone 4
Semi-
3.91 ± 0.44
0.15 ± 0.01

(SPV575)
Defined

EXP Clone 17
Semi-
4.30 ± 0.16
0.17 ± 0.02

(SPV578)
Defined

pTAL clones
Semi-
4.19 ± 0.16
0.18 ± 0.01

excluding Clone 9
Defined

pTAL Clone 9
Semi-
6.82 ± 1.11
0.22 ± 0.01

(SPV603)
Defined

The lipid profiles of all strains in the second (pTAL) full pathway screen were also quantified. For simplicity the 16 carbon fatty acid species are plotted in FIG. 35. As expected, Z11-16Acid is present only for strains expressing the desaturase. Complete lipid profiles were similar to those observed previously (FIG. 36). Z9-18Acid (oleic acid) was the second most abundant fatty acid species after Z11-16Acid.

Z9-16OH Functional Expression Assay

An in vivo, flask scale assay was used to test for Z9-16OH production. The parent control strain, H222 ΔPΔAΔF (SPV300), was compared to a strain expressing H. armigera FAR which relied on native Z9 desaturase activity to synthesize the Z9-16CoA precursor (SPV471). Biomass was generated through a YPD seed culture, mimicking the plate assay. Bioconversion flasks were inoculated at an initial OD600=1 or OD600=4 into the same Semi-Defined C:N=80 medium used in the Z11-16OH plate assay (See Materials & Methods for details). As expected, control flasks did not produce detectable Z9-16OH while SPV471 flasks produced up to 4.30±2.23 mg/L after 24 hours of incubation (FIG. 37A-FIG. 37B). While there was large variability between replicates, all SPV471 (H. armigera FAR) replicates exceeded 1 mg/L titer. Increased seeding density did not increase Z9-16Acid or Z9-16OH titer. The precursor Z9-16Acid titer at 24 hours was significantly less (<0.5 g/L) than the Z11-16Acid precursor observed for dual expression cassette strains used to produce Z11-16OH. The relative abundance of other fatty acid species was similar to previously observed profiles, with Z9-18Acid as the next most abundant species (FIG. 38). Both lipid and alcohol samples were taken over the course of 48 hours to produce a time course of Z9-16OH and lipid titers. Z9-16OH titer peaked at 24 hours before decreasing over the second day (FIG. 39A). Z9-16Acid increased rapidly over the first 24 hours before stabilizing or increasing slowly over the second 24 hours (FIG. 39B). Since the employed analytical method utilizes only the cell pellet, the decrease in Z9-16OH titer supports the hypothesis of downstream consumption or secretion of the alcohol products. They may be oxidized (co-oxidation), secreted as free alcohol, or derivatized and secreted as an ester. Analysis of supernatant samples using FID and MS SCAN detection revealed no detectable Z9-16OH or Z9-16OH derivatives supporting the hypothesis of consumption via oxidation pathways.

Conclusions

Combining expression of Helicoverpa Z11 desaturase and fatty acyl-CoA reductase led to production of Z11-16OH in Y. lipolytica H222 ΔPΔAΔF (SPV300) at titers >1 mg/L.

High C:N ratio conditions improved Z11-16OH titer relative to a rich medium condition.

Under lipid accumulating conditions the combination of native Z9 desaturase and H. armigera FAR activities are sufficient for synthesis of >1 mg/L Z9-16OH.

Titers are increased, for example, by deleting pathways consuming fatty alcohol products and/or fatty acid precursors; identifying FAR variants which exhibit higher turnover rate than H. armigera FAR; and/or increasing pathway copy number.

Key undesired byproducts are identified.

The possibility that some of the fatty alcohol product is converted into fatty acetate by the activity of one or more endogenous acetyltransferases is explored.

Improved host strains are engineered to eliminate the ω-oxidation pathway and components of the lipid storage pathway.

Materials & Methods

Strain Construction

All desaturase and reductase genes were ordered from Genscript. Homo sapiens codon optimization was used (Genscript algorithm). The newly synthesized expression cassette was subcloned into pPV199 by Genscript using the SapI restriction site. Plasmids were transformed and prepped from E. coli EPI400 using the Zyppy Plamsid Miniprep Kit (Zymo Research, Irvine, Calif.). Plasmids were digested with PmeI (New England Biolabs, Ipswich, Mass.) and purified by gel extraction using Zymoclean Gel DNA recovery Kit (Zymo Research, Irvine, Calif.). DNA was further concentrated using Clean and Concentrator Kit (Zymo Research, Irvine. Calif.). Approximately ˜1-2 μg of DNA was transformed using Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, Calif.). The manufacturer's protocol was modified as follows: A2 ml YPD seed culture was inoculated at 9 am the day before competent cell preparation. The seed was grown 8 hours (until 5 μm) before 40 ml of YPD in a 250 ml baffled shake flask (or 20 ml in a 125 ml baffled flask) was inoculated to an initial OD600 of 0.0005. The culture was incubated at 28° C. and 250 rpm ˜24 hours. Cells were harvested at an OD600=0.5-1. Instead of resuspending 10 ml of culture in 1 ml of Solution 2 as in the manufacturer's instructions (OD600-10), 10 ml of SPV140 culture was resuspended in 0.5 ml (OD600-20-30). All Solution 2 aliquots were slowly frozen to −80° C. by placing the tubes in a closed Styrofoam box before putting in the −80° C. freezer, 50 μl aliquots of competent cells in 1.7 ml Eppendorf tubes were thawed on ice, DNA eluted in water was added directly to the cells, and 500 μl of Solution 3 was used to suspend the cells with gentle pipetting. Tubes were incubated at 28° C. for 3 hours with gentle vortexing every 30 minutes. The entire transformation mixture was plated on CM glucose-ura agar plates. Positive integrants were found to be site-specific and genotyping was conducted by check PCR.

Z11-16OH Functional Expression Assay

Positive isolates were repatched onto YPD, grown overnight, and then stored at 4° C. Strains were inoculated from patch plates into 2 ml of YPD in 24 deepwell plates (square well, pyramid bottom). Replicate inoculations were made from each patch. Negative control strains were struck out on YPD from glycerol stocks and individual colonies were used to inoculate. Deepwell plates were incubated at 28° C. and 250 rpm in the Infors Multitron refrigerated flask shaker for 24 hrs. After 24 hrs of incubation, a 0.85 ml volume of each culture was pelleted by centrifugation at 800×g. Each pellet was resuspended in either 2 ml of YPD or Semi-defined medium (described in Table 17 below), 10 g/L methyl palmitate (pre-warmed to ˜50° C.) was added to cultures. All cultures were incubated for 48 hours before endpoint sampling. Final cell densities were measured with the Tecan Infinite 200pro plate reader, 1.5 ml (alcohol analysis) or 500 μl (lipid analysis) was transferred to 1.7 ml microcentrifuge tubes and pelleted. Supernatant was transferred to clean tubes and samples were processed as described below.

TABLE 17

Semi-defined (C:N = 80) medium composition. Components of the

semi-defined base medium used to induce lipid storage are described.

Media Components
Conc.
Units

Yeast Extract
2
g/L

Peptone
1
g/L

Potassium phosphate buffer pH7
0.1
M

YNB w/o aa, NH4
1.7
g/L

Glucose
60
g/L

Glycerol
5
g/L

Z9-16OH Functional Expression Assay

SPV300 (negative control) and SPV471 were struck out onto YPD agar plates, grown overnight, and then stored at 4° C. Strains were inoculated from colonies into 2 ml of YPD and incubated at 28° C. and 250 rpm in 14 ml round bottom culture tubes for ˜8 hours. After incubation, 2 ml of culture was used to inoculate 20 ml of YPD in a 125 ml baffled shake flask. Shake flasks were incubated 24 hrs at 28° C. and 250 rpm. After incubation, cell density in shake flasks was measured using a Tecan Infinite 200pro plate reader. An appropriate volume of culture was pelleted in order to resuspend cells in 25 ml of Semi-defined C:N=80 medium (see Table 17 above) at an initial OD600=1 (˜1 gDCW/L) or 4 (˜4gDCW/L). The resuspended culture was added to 250 ml baffled shake flasks. Neat methyl palmitate was added at 10 g/L final concentration after pre-heating to 50° C. After substrate addition, flasks were incubated at 28° C. and 250 rpm for two days. At 12, 18, 24, 36, 42, and 48 hours 500 μl (lipid analysis) and 1.5 ml (alcohol analysis) samples were taken in 1.7 ml microcentrifuge tubes. Samples were pelleted and the supernatant was transferred to a clean microcentrifuge tube.

Metabolite Extraction and GC-MS Detection

Alcohol Analysis

The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL crimp vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 μL 2.5 N HCl the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM methyl heptadecanoate were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase were transferred into a GC vial. The organic phase was evaporated in a heat block at 90° C. for 30 min. The residue was dissolved in 50 μL N,O-Bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane. Prior to transfer into glass inserts the mixture was heated 5 min at 90° C. The samples were analyzed by GC-MS (Table 18).

TABLE 18

GC-MS parameters

System
Agilent 6890 N GC, ChemStation G1701EA E.02.01.1177

Column
DB23 30 m × 25 μm × 25 μm

Pressure = 11.60 psi; Flow = 0.6 mL/min

Inlet
Heater = 250° C.; Pressure = 11.74 psi;

Total Flow {He} = 111 mL/min

Carrier
He @ 29 cm/sec, 11.60 psi

Signal
Data rate = 2 Hz/0.1 min

Oven
150° C. for 1 min

Ramp 12° C./min to 220° C., hold 3 min

Ramp 35° C./min to 300° C., hold 4 min

Injection
Splitless, 250° C.

Detector
Initial strain screening and first technical triplicate: HP 5973

MSD in SIM mode (m/z: 208.0, 297.3 and 387.3),

SPV488/SPV490 alcohol quantification: HP 5973 MSD in SIM

mode (m/z: 284.0 and 297.3),

100 msec Dwell, EMV mode: Gain factor 1,

2.4 min solvent delay, 3.09 cycles/sec

Sample
Injection volume = 1 uL

Lipid Analysis

Total lipid composition was based on modified procedures by Moss et al. (1982) and Yousuf et al (2010). The pelleted cells (in 1.5 mL plastic tubes), usually about 10 mg to 80 mg, were resuspended in methanol containing 5% (w/w) of sodium hydroxide. The alkaline cell suspension was transferred into a 1.8 mL glass crimp GC-vial. The mixture was heated for 1 h in the heat block at 90° C. Prior to acidification with 400 μL 2.5 N HCl, the vial was allowed to cool to room temperature. 500 μL chloroform containing 1 mM methyl heptadecanoate were added and the mixture was shaken vigorously, then both aqueous and organic phase were transferred into a 1.5 mL plastic tube. The mixture was centrifuged at 13,000 rpm, afterwards 450 μL of the organic phase was transferred into a new 1.8 mL glass screw-cap GC-vial. After cooling to room temperature residual fatty acid methyl esters and free fatty acids were dissolved and derivatized in methanol containing 0.2 M TMSH (trimethylsulfonium hydroxide)(Table 19).

TABLE 19

GC-MS parameters

System
Agilent 6890 GC, ChemStation Rev. B.03.02 (341)

Column
J&W DR-23 30 m × 25 mm × 25 μm

Pressure = 16 psi; Flow = 0.9 mL/min; Run Time = 14.4 min

Inlet
Heater = 240° C.; Pressure = 16 psi; Total Flow {He} = 31.4

mL/min

Carrier
H₂@ 1 mL/min, 9 psi, 35 cm/sec

Signal
Data rate = 2 Hz/0.1 min

Oven
150° C. for 1 min

Ramp 12° C./min to 220° C., hold 3 min

Ramp 35° C./min to 240° C., hold 6 min

Equilibration Time: 2 min

Injection
Split, 240° C.

Split ratio - 30:1, 29.1 mL/min

Detector
FID, 240° C.

H₂@ 35.0 mL/min, Air @ 350 mL/min;; Electrometer

{Lit Offset} @ 2.0 pA

Sample
Injection volume = 1 uL

Example 10: Production of Z11-14Acid in Yarrowia lipolytica

Background and Rationale

Yarrowia lipolytica was engineered to produce Z11-14Acid, the precursor to target Lepidoptera pheromone Z11-14Ac.

A library of 73 desaturases was chosen to target potential pheromones including Z11-14Ac, Z7-12Ac, Z9E12-14Ac, E8E10-C12OH and Z9E11-14Ac. All desaturases were tested in the H222 ΔPΔAΔF (SPV300) background.

Eleven desaturases were identified from literature to have Δ11 activity (DST001-DST009, DST030, and DST039, Table 20). All desaturases were screened by feeding either methyl palmitate (C16), methyl myristate (C14), or methyl laurate (C12) as substrate, and full product profiles were determined by GC analysis.

The resulting activity of the purported Δ11 desaturase library, and other desaturases shown to produce Δ11 compounds, specifically Z11-14Acid, is discussed.

TABLE 20

Desaturases discussed in Example 10

Enzyme

GenBank

Code
Organism of origin
Accession

DST001

Argyrotaenia
velutinana

AF416738

DST002

Spodoptera
litura

AGH12217.1

DST003

Sesamia
inferens

AII21943.1

DST004

Manduca
sexta

CAJ43430.2

DST005

Ostrinia
nubilalis

AF441221

DST006

Helicoverpa
zea

AAF81787.1

DST007

Choristoneura
rosaceana

AF545481

DST008

Drosophila
melanogaster

AJ271414

DST009

Spodoptera
littoralis

AY362879

DST030

Lampronia
capitella

ABX71630.1

DST039

Amyelois
transitella

NP001299594.1

Results

Up to 69 mg/L Z11-14Acid production was observed when feeding 2 g/L methyl myristate to the desaturase library (FIG. 41). The current best desaturase, Helicoverpa zea (Hz) DST (SPV459, encoded by SEQ ID NO: 54), in addition to desaturases DST001 through DST007, DST030 and DST039, produce some amount of Z11-14Acid ranging from 16 mg/L to 69 mg/L. DST001 (A. velutinana), DST004 (A. sexta), and DST039 (A. transitella) are more specific for Z11-14Acid production than Z11-16Acid production, although these desaturases produce ˜20 mg/L Z11-14Acid. Strains producing higher Z11-14Acid titer also produced Z9-14Acid from the methyl myristate substrate at 20-30 mg/L, which was reduced compared to the negative control SPV298. The C14-C18 product profile of Hz DST (SPV459) compared to SPV298 is shown in FIG. 42.

Proof-of-concept of Z11-14Acid synthesis is shown. Attempts were made to identify enzymes that had improved Z11-16Acid titer or product specificity over Helicoverpa zea DST (1.05 g/L Z11-16Acid; 69 mg/L Z11-14Acid). While there were no desaturases that had higher production than Hz DST (SPV459), DST003 (SEQ ID NO: 39) had similar production phenotypes to the HzDesat strain, and DST002 and DST005 had similar Z11-16Acid production with reduced Z11-14Acid. The desaturase in DST006 is genetically equivalent to the H. zea desaturase expressed in SPV459 and served as a library control. DST006 produced equivalent levels of Z11-16Acid when fed methyl palmitate; however this strain produced a lower titer of Z11-14Acid on methyl myristate. Genetic variation in strain background may account for the observed difference.

DST039 (A. transitella) was previously screened under different conditions. In rich media, Z11-16Acid production with the native A. transitella coding sequence was not observed. The H. sapiens optimized sequence was tested and still no activity was observed with the rich medium condition. In this screen, DST039 was tested in nitrogen limited condition with Hs optimized sequence and resulted in 235 mg/L production of Z11-16Acid and 21 mg/L Z11-14Acid on the relevant substrates.

Δ11 products from DST008 (Drosophila melanogaster) or DST009 (Spodoptera littoralis) in the SPV300 background were not observed.

Summary

Z11-14Acid production was observed in ten desaturases with titers ranging from 16 mg/L to 69 mg/L (2 g/L methyl myristate fed).

H. zea DST (SEQ ID NO: 54) remained the best Z11-16Acid producer (>1 g/L when fed with methyl palmitate).

DST003 (S. inferens, SEQ ID NO: 39) has the most similar phenotype to H. zea DST.

DST002 (S. litura) and DST005 (O. nubialis) are more specific than H. zea DST for Z11-16Acid production (reduced Z11-14Acid production).

DST001 (A. velutinana). DST004 (M. sexta), and DST039 (A. transitella) are more specific than H. zea DST for Z11-14Acid production.

Conclusions

Z11-14Acid can be produced with the heterologous expression of specific desaturases in Yarrowia lipolytica when feeding methyl myristate.

Multiple copies of desaturase (identical or combination of sequences) are integrated in improved strain backgrounds for increased Z11-14Acid titer, product specificity, and genetic stability.

Materials & Methods

Library Generation

Desaturase sequences were provided to Genscript for codon optimization (Homo sapiens expression organism) cloning into pPV266 (XPR2 locus integration vector with TEF promoter and terminator) using PacI/SapI restriction digestion. Lyophilized DNA was provided as well as EPI400 agar stabs. Desaturase constructs are listed in Table 21.

Constructs were linearized using PmeI restriction enzyme and directly transformed into host strain SPV300. Transformants were verified by check PCR using primers outside of the XPR2 integration junction and within the pTEF promoter.

TABLE 21

Desaturase constructs

Enzyme

GenBank

E.
coli

Plasmid

Code
Species
Accession
SPV
pPV

DST001

Argyrotaenia

AF416738
SPV0609
pPV0300

valutinana

DST002

Spodoptera

AGH12217.1
SPV0610
pPV0301

litura

DST003

Sesamia

AII21943.1
SPV0611
pPV0302

inferens

DST004

Manduca

CAJ43430.2
SPV0612
pPV0303

sexta

DST005

Ostrinia

AF441221
SPV0613
pPV0304

nubilialis

DST006

Helicoverpa

AAF81787.1
SPV0614
pPV0305

zea

DST007

Choristoneura

AF545481
SPV0615
pPV0306

rosaceana

DST008

Drosophila

AJ271414
SPV0616
pPV0307

melanogaster

DST009

Spodoptera

AY362879
SPV0617
pPV0308

littoralis

DST030

Lampronia

ABX71630.1
SPV0638
pPV0329

capitella

DST039

Amyelois

NP_001299594. 1
SPV0647
pPV0338

transitella

Plasmid Digest

˜10 μg of lyophilized DNA was ordered from Genscript. DNA was resuspended in 50 μL water for a final concentration of ˜200 ng/L, 10 μL of DNA was mixed with 1.25 μL 10× CutSmart Buffer and 1.25 μL PmeI restriction enzyme (12.5 μL reaction volume). The reaction was incubated in the PCR machine for 1.5 hours at 37° C. and heat inactivated at 65° C. for 30 minutes.

Transformation

SPV300 competent cells were grown by inoculating a YPD culture at 0.001 OD in a baffled flask and growing until 0.5-1.0 OD. Cells were harvested at 800×g and washed with 0.25× volume of Solution 1 from the Zymo Frozen-EZ Transformation II Kit for Yeast. Cells were resuspended in Solution 2 at 1000× concentration of the original culture volume and slowly frozen at −80° C. while insulated in a styrofoam container (frozen cells may have better transformation efficiency over fresh), 50 μL of cells were first mixed with the 12.5 μL digestion reaction (no cleanup necessary), and then with 500 μL Solution 3. Transformations were incubated for 3 hours at 28° C. without shaking, after which the full transformation mixture was plated to appropriate selective agar media. Petri dishes were incubated for 3-4 days before the appearance of colonies.

Check PCR

Transformation colonies were picked to 7 μL water in a PCR plate, 5 μL of cells were patched by multichannel to selective omni trays and grown overnight. The remaining 2 μL of cells were microwaved for 2 minutes before adding 15 μL of PCR master mix.

PCR Master Mix
1× reaction

2× Phusion Master Mix (HF Buffer)
7.5 μL

100 μM oPV204 (XPR2 locus F)
0.1 μL

100 μM oPV195 (pTER R)
0.1 μL

Water
7.3 μL

PCR Cycle:

Temp.
Time
Cycles

98° C.
2
min
1×

98° C.
15
sec
30×

64° C.
30
sec

72° C.
60
sec

72° C.
5
min
1×

4° C.
∞
1×

Colony Patching

Positive clones were re-patched to YPD omni trays in 24-well format including assay controls. Omni trays were grown overnight at 28° C. and used to inoculate bioassay cultures.

Bioassay

Positive transformants (N=4 clones per construct) were inoculated into 1 mL YPD in a 24-well culture plate and incubated for 24 hours in the Infors HT Mulitron Pro at 28° C. with 1000 rpm shaking. Cells were pelleted at 800×g and resuspended in S2 media with 5 μL substrate (˜2 g/L concentration), 250 μL of culture was sampled into glass crimp top vials after 48 hours of bioconversion.

S2 Media

2 g/L Yeast Extract, 1 g/L Peptone, 0.1M Phosphate buffer, 1.7 g/L YNB w/o aa, NH4, 60 g/L Glucose, 5 g/L Glycerol

GC Sample Processing

Front Inlet/Detector:

System
6890 GC, ChemStation Rev. B.03.02 (341)

Column
J&W DB-23 30 m × 25 mm × 25 um

Run Time = 14.4 min

Inlet
Heater = 240° C.; Pressure = 9.0 psi; Total Flow {H2} = 36.2

mL/min

Carrier
H2 @ 1.0 mL/min, 9.0 psi, 35 cm/sec

Signal
Data rate = 2 Hz/0.1 min

Oven
150° C. for 1 min

Ramp 12° C./min to 220° C., hold 3 min

Ramp 35° C./min to 240° C., hold 4 min

Equilibration Time: 2 mim

Injection
Split, 240° C.

Split ratio - 30:1; 29.1 mL/min

Detector
FID, 240° C.

H2 @ 35.0 mL/min, Air @ 350 mL/min

Electrometer {Lit Offset} @ 2.0 pA

Sample
Injection volume = 1 μL

Back Inlet/Detector:

System
6890 GC, ChemStation Rev. B.03.02 (341)

Column
J&W DR-23 30 m × 25 mm × 25 um

Run Time = 14.4 min

Inlet
Heater = 240° C.; Pressure = 9.8 psi; Total Flow {H2} = 40.1

mL/min

Carrier
H2 @ 1.1 mL/min, 9.8 psi, 38 cm/sec

Signal
Data rate = 2 Hz/0.1 min

Oven
150° C. for 1 min

Ramp 12° C./min to 220° C., hold 3 min

Ramp 35° C./min to 240° C., hold 4 min

Equilibration Time: 2 min

Injection
Split, 240° C.

Split ratio - 30:1; 32.3 mL/min

Detector
FID, 240° C.

H2 @ 35.0 mL/min, Air @ 350 mL/min

Electrometer {Lit Offset} @ 2.0 pA

Sample
Injection volume = 1 μL

TMSH: Trimethylsulfonium Hydroxide (0.2 mol/L in Methanol)—VWR TCT1576-025ML

Example 11: Production of Z11-16OH in Yarrowia lipolytica Using Increased Copy Number of or Engineered Variant Fatty Alcohol Forming Fatty Acyl-CoA Reductases (FARs)

Background and Rationale

Engineering the microbial production of unsaturated insect fatty alcohols requires the functional expression of a synthetic pathway. One such pathway comprises a transmembrane desaturase to mediate the conversion of fatty acyl-CoA into regio- and stereospecific unsaturated fatty acyl-CoA. An alcohol-forming reductase (FAR) complements the synthetic pathway to produce the respective fatty alcohol.

In some insects species the respective FAR enzymes are activated via site specific dephosphorylation (Jurenka, R. & Rafaeli, A. Regulatory Role of PBAN in Sex Pheromone Biosynthesis of Heliothine Moths. Front. Endocrinol. (Lausanne), 2, 46 (2011); Gilbert, L. I. Insect Endocrinology. (Academic Press)). Phosphorylation of heterologously expressed FAR enzymes in Y. lipolytica may lead to inactivation, and result in low fatty alcohol titers. A bioinformatic approach was used to predict phosphorylated residues within HaFAR.

Alanine substitution of serine and threonine residues has been shown to abolish phosphorylation (Shi, S., Chen, Y., Siewers, V. & Nielsen, J. Improving Production of Malonyl Coenzyme A-Derived Metabolites by Abolishing Snf1-Dependent Regulation of Acc1, mBio 5, (2014)). Thus, in addition to increasing FAR gene copy number, the impact of alanine substitutions of several serine residues of an HaFAR enzyme (HaFAR amino acid sequence set forth in SEQ ID NO: 41 and HaFAR nucleotide sequence set forth in SEQ ID NO: 90) on Z11-16OH titer was tested.

Approach

A second copy of human codon-optimized H. armigera FAR gene (HaFAR) was introduced into the chromosome of the Z11-16OH producer parent strain. In parallel, the effects of introducing a copy of a mutated H. armigera FAR gene variant towards Z11-16OH production improvement were also explored. Seven mutated variants were designed with the aim to increase FAR activity by potentially relieving the requirement for dephosphorylation (as observed in some insect species). To this end, several potential phosphorylation sites within the amino acid sequence of the H. armigera FAR were identified, and replaced with alanine.

Results

Determination of Phosphorylated Sites in HaFAR

The server at world wide web address: cbs.dtu.dk/services/NetPhos/( ) predicts potential phosphorylation sites based on a database (world wide web address: phospho.elm.eu.org/about.htmL) of experimentally verified phosphoproteins based on 17 kinases: ATM, CKI, CKII, CaMII, DNAPK, EGFR, GSK3, INSR, PKA, PKB, PKC, RSK, SRC, cdc2, cdk5 and p38MAPK.

The software program predicted 22 serine, 11 threonine and 10 tyrosine as potential phosphorylation sites in H. armigera FAR (FIG. 43).

Next, a tailored prediction program to determine phosphorylation sites of the H. armigera FAR upon expression in yeasts was applied (world wide web address: cbs.dtu.dk/services/NetPhosYeast/, Blom, N., Gammeltoft, S. & Brunak, S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites1. J. Mol. Biol. 294, 1351-1362 (1999)). Since in yeast no tyrosine kinases have been identified yet (Ingrell, C. R., Miller. M. L., Jensen. O. N. & Blom. N. NetPhosYeast: prediction of protein phosphorylation sites in yeast. Bioinforma. 23, 895-897 (2007)), the yeast-specific software only considers serine and threonine as potential phosphorylation sites. It was striking that both programs predicted the same 11 serine residues to be phosphorylated. In contrast, the yeast specific analysis tool did not predict any phosphorylated threonine residues (FIG. 44).

In a first experiment a small library consisting of 7 Ser to Ala point mutants was tested (Table 22). Three predicted phosphorylation sites were not considered for mutagenesis (position Ser301, Ser386. Ser416 scored barely above the threshold). For the remaining 7 serine residues, alanine substitutions were introduced.

TABLE 22

Serine to alanine mutant library

Additional

Parent strain
Enzyme
Strain #

Y. lipolytica H222ΔPΔAΔF
HaFAR
SPV916

xpr2::pTEF-HZ_Z11_desat_

Hs-tLIP2-pTAL1-HA_FAR-

tXPR2_loxP

Y. lipolytica H222ΔPΔAΔF
HaFAR S60A
SPV909

xpr2::pTEF-HZ_Z11_desat_

Hs-tLIP2-pTAL1-HA_FAR-

tXPR2_loxP

Y. lipolytica H222ΔPΔAΔF
HaFAR S195A
SPV910

xpr2::pTEF-HZ_Z11_desat_

Hs-tLIP2-pTAL1-HA_FAR-

tXPR2_loxP

Y. lipolytica H222ΔPΔAΔF
HaFAR S298A
SPV911

xpr2::pTEF-HZ_Z11_desat_

Hs-tLIP2-pTAL1-HA_FAR-

tXPR2_loxP

Y. lipolytica H222ΔPΔAΔF
HaFAR S378A
SPV912

xpr2::pTEF-HZ_Z11_desat_

Hs-tLIP2-pTAL1-HA_FAR-

tXPR2_loxP

Y. lipolytica H222ΔPΔAΔF
HaFAR S394A
SPV913

xpr2::pTEF-HZ_Z11_desat_

Hs-tLIP2-pTAL1-HA_FAR-

tXPR2_loxP

Y. lipolytica H222ΔPΔAΔF
HaFAR S418A
SPV914

xpr2::pTEF-HZ_Z11_desat_

Hs-tLIP2-pTAL1-HA_FAR-

tXPR2_loxP

Y. lipolytica H222ΔPΔAΔF
HaFAR S453A
SPV915

xpr2::pTEF-HZ_Z11_desat_

Hs-tLIP2-pTAL1-HA_FAR-

tXPR2_loxP

The HaFAR library was custom synthesized, and subcloned into plasmid pPV234 for expression under TEF promoter, and XPR2 terminator at the AXP locus. Linearized constructs were transformed into strain Y. lipolytica H222ΔPΔAΔF SPV603 which expresses H. zea Z11 desaturase (SEQ ID NO: 54) combined with the H. armigera FAR (SEQ ID NO: 41). This approach determined the impact of a second copy e.g. protein expression in addition to the impact of the individual point mutations.

Four individual clones of positive integrants were tested. Cultivation was performed in 24 well plates. Briefly, 2 mL YPD was inoculated from patches of individual clones and incubated for 24h at 28° C. 1000 rpm. After 24h OD600 was measured and the cells were centrifuged at 1000 rpm (Table 23). The cell pellets were resuspended in 1 mL S2 media and 10 g/L methyl palmitate was added. The cells were incubated for 18h at 28° C., 1000 rpm. Cultivations were stored at −20° C. until analyzed. Extraction and analysis was performed according to previously established standard protocols using GC-FID.

TABLE 23

OD600 measurement of the 24 well plate cultivations (two plates were cultivated

in parallel). Each individual well was measured as duplicates upon 1:10 dilution after 24 h

cultivation in YPD. Calculated OD600 values are given.

HaFAR
HaFAR
HaFAR
HaFAR

HaFAR-
HaFAR-
HaFAR-
HaFAR
S195A
S195A
S195A
S195A
HaFAR
HaFAR

GFP
GFP
GFP
S60A
Clone 2
Clone 2
Clone 3
Clone 3
S298A
S298A
SPV603
SPV603

OD600
1
2
3
4
5
6
7
8
9
10
11
12

A
15.392
16.16
17.376
17.976
17.56
18.272
14.188
14.774
14.774
15.708
17.14
17.24
plate2

B
16.62
17.172
18.632
18.596
18.894
18.088
16.082
15.38
16.04
16.03
16.572
16.584
plate2

C
16.978
16.952
18.154
18.224
18.678
19.384
16.062
14.858
15.498
15.58
18.14
17.528
plate2

D
16.596
17.23
17.668
18.842
18.39
18.792
20.678
16.566
16.094
15.904
1.262
1.152
plate2

E
17.53
17.156
16.78
15.776
17.398
16.736
16.154
15.514
16.748
16.792
18.26
17.322
plate1

F
18.336
17.928
16.392
15.33
20.018
19.37
15.06
14.266
17.522
17.076
16.724
16.522
plate1

G
17.16
17.224
15.984
16.194
17.878
17.626
15.372
15.15
16.688
17.022
17.742
16.942
plate1

H
16.258
16.12
16.134
16.236
16.808
16.176
15.382
14.51
15.456
15.394
7.664
7.956
plate1

HaFAR
HaFAR
HaFAR
HaFAR
HaFAR
HaFAR
HaFAR
HaFAR

S378A
S378A
S394A
S394A
S418A
S418A
S453A
S453A
HaFAR
HaFAR
SPV603
SPV603

OD600
1
2
3
4
5
6
7
8
9
10
11
12

A
15.842
15.97
15.31
16.278
16.088
16.302
16.074
15.502
15.34
15.742
16.89
16.062
plate 2

B
16.3
17.306
16.064
16.5
16.64
16.344
15.69
15.598
15.434
15.248
16.674
16.476
plate 2

C
17.088
17.146
15.966
15.472
16.508
16.548
15.82
15.216
15.796
16.098
16.81
17.246
plate 2

D
17.512
17.198
15.15
14.168
16.094
13.93
15.642
15.078
16.77
16.242
0.9
0.864
plate 2

E
16.446
17.014
15.762
16.536
17.188
17.372
16.164
15.892
15.8
16.422
17.998
17.814
plate 1

F
16.982
11.196
16.098
13.148
16.1
12.348
16.688
10.232
15.596
13.068
18.456
16.098
plate 1

G
15.4
15.568
15.236
16.348
15.504
15.824
14.922
15.622
15.11
15.576
17.142
17.672
plate 1

H
17.088
16.63
0.968
1.006
13.82
12.49
15.826
15.27
17.132
16.166
1.082
0.932
plate 1

As shown in FIG. 45, strains expressing a second HaFAR copy encoding a point mutation, HaFAR-GFP or HaFAR* (where * indicates a second copy of the parental HaFAR enzyme in addition to the existing copy of the parental strain). The copy increased fatty alcohol titers when compared to the parental strain (SPV603). The introduction of HaFAR (S60A), HaFAR (S298A), HaFAR (S394A), HaFAR (S453A) was neutral or showed slight increases when compared to the HaFAR double copy strain. In contrast, expression of HaFAR (S418A), and HaFAR (S195A) resulted in a distinct increase of Z11-16OH titers.

Overall, these results suggest that residue 195 and 418 are important for increasing HaFAR activity in Y. lipolytica. Their substitutions to alanine may inhibit phosphorylation; therefore, a dephosphorylation mechanism was not required for their enhanced activity. Among other explanations, the activating effect of the serine to alanine mutation could be from improved protein folding, increased stability or higher protein expression.

In addition, several fatty acid species were quantified to determine whether the increased Z11-16OH production led to Z11-16Acid depletion. The standard deviations for some samples were very high (FIG. 46). In all samples saturated and Z11-16Acid was detected. Quantification of the respective product intermediate Z11-16Acid showed increased consumption for the mutant strains SPV910 and SPV914 expressing HaFAR S195A and HaFAR S418A.

Chromosomal integration of an additional HaFAR into the parent strain increased the Z11-16OH titer from ˜20 mg/L to 40 mg/L in shake flask experiments.

When HaFAR (Ser195Ala) or HaFAR (Ser418Ala) was introduced, Z11-16OH titer increased from ˜40 mg/L to ˜120 mg/L or ˜80 mg/L, respectively in shake flask experiments.

The pathway intermediate Z11-16Acid for mutant strains SPV910 harboring HaFAR (Ser195Ala), and SPV914 harboring HaFAR (Ser418Ala) accumulated at ˜100 mg/L less than the parent strain SPV603.

Conclusions

The addition of another H. armigera FAR gene into Z11-16OH producer strain marginally increased titer. Introduction of mutated FARs, however, significantly improved Z11-16OH by up to ˜7×. This suggests that Y. lipolytica phosphorylates FAR enzymes, and that FAR dephosphorylation is a bottleneck for its full activity. The designed mutations in HaFAR may relieve its requirement for dephosphorylation to convert Z11-16Acid into Z11-16OH. It is also possible that the designed mutations improved FAR activity through a dephosphorylation-independent mechanism. Shake flask experiments suggest a direct correlation between biomass, time and fatty alcohol titers.

Second generation strains are created for further improvement in Z11-16OH by, for example, eliminating lipid storage pathways (e.g. diacylglycerol acetyltransferase (DGAT) gene deletions), and/or eliminating byproduct (hydroxyacid, diacid) pathways.

Materials & Methods

Marker rescue of SPV603

30 mL of CM minus uracil were inoculated with SPV603 in a 250 mL baffled shake flask. The culture was incubated in a bench top shaker at 250-300 rpm and 28° C. The following morning the OD600 of the culture was measured. Cells were harvested at OD600: 0.6. Cells were pelleted in a falcon tube (800×g, 5 min). Supernatant was removed and wash in ½ volume (15 mL) of Solution 1 from the Zymo kit. Cells were pelleted again (800×g, 5 min) and resuspended in 200 μl of Solution 2. A 50 μl aliquot was used directly in a 1.7 mL Eppendorf tube. Remaining aliquots were stored at −80° C. 10 μl of DNA (1-2 μg) was added and gently mixed with cells. 500 μl of Solution 3 was added and mixed gently. Transformation mix was incubated for ≥3 hours in 28° C. plate incubator subsequently washed with 2 mL deionized water (autoclaved). Recovery was performed in 2 mL YPD o/n at 28° C. The transformation mix was plated directly on selection plate. Clones were picked and repatched on URA minus FOA 0.1% and YPD plates. URA removal was confirmed using standard PCR protocols.

Construction of HaFAR Integration Cassette

The HaFAR library was custom synthesized with human codons (Genscript), and subcloned into plasmid pPV234 using the restriction site SpeI and NotI for expression under TEF promoter, and XPR2 terminator at the AXP locus. Linearized constructs were transformed into strain Y. lipolytica H222ΔPΔΔF SPV603 which expresses H. zea Z11 desaturase (SEQ ID NO: 54) combined with the H. armigera FAR (SEQ ID NO: 41). Amino acid sequences for the variant FARs are set forth in SEQ ID NOs: 42-48. Nucleotide sequences for the variant FARs are set forth in SEQ ID NOs: 83-89.

Cultivation of HaFAR Mutant Library in 24 Well Plates

In a first round four individual isolate from each transformation was tested. Cultivation was performed in 24 well plates. 2 mL YPD was inoculated from patches of individual clones and incubated for 24h at 28° C., 1000 rpm. After 24h OD600 was measured and the cells were centrifuged at 1000 rpm. The cell pellets were resuspended in 1 mL S2 media and 10 g/L methyl palmitate was added. The cells were incubated for 24h at 28° C., 1000 rpm. Cultivations were stored at −20° C. until analyzed. Next, four clones of the best performing positive integrant were tested. Cultivation was performed in 24 well plates. 2 mL YPD was inoculated from patches of individual clones and incubated for ˜28h at 28° C. 1000 rpm. After 28h OD600 was measured and the cells were centrifuged at 1000 rpm. The cell pellets were resuspended in 1 mL S2 media and 10 g/L methyl palmitate was added. The cells were incubated for 18h at 28° C., 1000 rpm. Cultivations were stored at −20° C. until analyzed.

Cultivation of HaFAR Mutant Library in Shake Flasks

Shake Flask Experiment 1: Time-Course Analysis of Fatty Alcohol and Fatty Acid Titers During Shake Flask Cultivation

In a first step a shake flask experiment of the respective strains was performed to analyze fatty alcohol and fatty acid formation over time. The best performing clone of the strains expressing HaFAR. HaFAR S195A and HaFAR S418A were tested. 10 mL YPD was inoculated from patches of individual clones and incubated for 24h at 28° C., 250 rpm in a 125 mL shake flask. Next, 25 mL YPD were inoculated with 5 mL starter culture in 500 mL shake flasks and incubated for 24h at 28° C., 250 rpm. The cells were harvested and the pellets were resuspended in 15 mL S2 media and 10 g/L methyl palmitate was added. OD600 was measured and subsequently methyl palmitate was added. The cells were incubated for 72h at 28° C., 1000 rpm. 4×0.5 mL samples were taken every 24h and stored at −20° C. until analyzed according to a previously established standard protocol (FIG. 47 and FIG. 48). To improve sampling and reduce the standard deviation, samples were transferred directly into GC crimp vials and stored until analyzed.

The OD600 for the strains were: SPV910-10.2, SPV914-11.5 and SPV916-12.3.

Shake Flask Experiment 2: Impact of Increased Biomass on Bioconversion

The time-course experiment revealed a decrease in fatty alcohols over time. Thus, sampling was reduced to 20h. In previous 24 well plate bioconversion experiments the measured biomass was ˜2× higher compared to the shake flask experiment. Next a shake flask experiment of the respective strains was performed to analyze the impact of biomass on fatty alcohol titers. The overall incubation time was reduced and the biomass was increased during bioconversion phase. The best performing clone of the strains expressing HaFAR S195A and HaFAR S418A were tested. 20 mL YPD was inoculated from patches of individual clones and incubated for 28h at 28° C., 250 rpm in a 500 mL shake flask. The cells were harvested and the pellets were resuspended in 10 mL S2 media and 10 g/L, OD600 was measured and subsequently methyl palmitate was added. The cells were incubated for 20h at 28° C., 1000 rpm. 4×0.5 mL samples were taken and analyzed according to a previously established standard protocol (FIG. 49).

The OD600 for the strains were: SPV910-24.9 and SPV914-24.7.

Metabolite Extraction

Cell pellets were resuspended with 500 μL 5% NaOH (in methanol) then heated for 1 hour at 85° C. Samples were cooled down and then acidified by adding 400 μL 5N HCl. Next, 500 μL chloroform (containing 1 mM C17:0 heptadecanoate internal standard) were added to samples. Samples were mixed vigorously then spun down for 2 minutes at 13,000 RPM using a table top centrifuge. 450 μL chloroform were transferred to a new vial then evaporated at 85° C. for ˜15 minutes. Samples were then resuspended in 50 μL BSTFA (and were now ready for GC analysis). After analysis of the fatty alcohols samples were diluted ˜1:10 and rerun on the GC to improve peak separation of the fatty acid peaks.

Quantification

All quantifications were based on the concentration of the internal standard. The concentration of the internal standard C₁₇ME is 1 mM. The final concentration of fatty alcohols as well as fatty acids were calculated based on the effective carbon number compared to the internal standard.

Example 12: Production of Fatty Alcohol and Fatty Acid in Yarrowia lipolytica Using Second Generation Strains Engineered to Eliminate Lipid Storage Pathways or Using Additional Variant Fatty Alcohol Forming Fatty Acyl-CoA Reductases (FARs)

The impact of an endogenous diacyl glycerol acyltransferase deletion selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2) was tested. Each dga gene was deleted individually in the strain SPV735 (ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP). Subsequently, the individual selection marker in each deletion strain SPV957 (Δdga1, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) and SPV959 (Δdga2 ΔURA, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) according to standard procedures was performed. The resulting strain SPV1053 (Δdga1 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) and SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) were used to transform HaFAR and HaFARS195A. The formation of fatty alcohols compared to the strain SPV603 was tested in shake flasks (FIG. 50 and FIG. 51). The results in shake flasks over time suggest that the deletion of each individual dga gene improves fatty alcohol formation and decreases fatty acid storage.

Time-Course Analysis of Fatty Alcohol and Fatty Acid Titers During Shake Flask Cultivation

A copy of the enzyme HAFAR (SEQ ID NO: 41) or HaS195A (SEQ ID NO: 43) was introduced into the strain SPV1053 (Δdga1 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) and SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP), each of which expresses H. zea Z11 desaturase (SEQ ID NO: 54) and is deleted for either DGA1 or DGA2 diacylglycerol acyltransferase.

Cultivation was performed as biological triplicates in shake flasks. The starting culture for each strain was grown in 50 mL YPD in 250 mL shake flask for 28h at 250 rpm, 28° C. Cells were harvested via centrifugation at 800 g for 5 min at room temperature and resuspended in 30 mL S2 media. Cell suspensions were normalized to OD600: ˜8. Each starting culture was split in three individual 125 mL shake flasks. 10 g/L methyl palmitate was added. Cultures were incubated for 72h. Sampling was performed every 24h and OD600 for each shake flask was measured. FIG. 50 and FIG. 51 show fatty alcohol titers and fatty acid titers, respectively, for the time course experiments.

Extraction of cultivation samples. 0.5 mL of each sample were harvested directly into crimp vials. The crimp vials were centrifuged at 800 g for 5 min. The supernatant was removed and the samples were sealed and stored at −20° C. until analysis. Samples were resuspended in 500 μL methanol containing 5% KOH and incubated for 60 min at 65° C. to 85° C. 200 μL 5N HCL was added to each crimp vial after cool down step. 600 μL chloroform containing 1 mM C17Me were added and sealed again. Samples were mixed and centrifuged. 500 uL of the chloroform was transferred to a new GC vial and dried to completeness at 85° C. for 30 min. Samples were resuspend in 100 uL BSTFA and incubated at room temperature for 1 h. Samples were analyzed according to standard protocol using gas chromatography.

Fatty Alcohol Forming Fatty Acyl-CoA Reductase Library Screening of New Strains in 24 Well Plates

A single copy of each respective FAR enzyme from Table 24 was introduced into the strain SPV1054 (Δdga2 ΔURA, ΔLeu, leu2::pTEF-HZ_Z11_desat_Hs-tXPR2_loxP) expressing H. zea Z1 desaturase (SEQ ID NO: 54) and deleted for DGA2 diacylglycerol acyltransferase.

Cultivation was performed as biological quadruplicates in 24 well plates. 2 mL YPD were inoculated from a patch of each individual transformant. 24 well plates were incubated for 28h at 250 rpm, 28° C. Cells were harvested via centrifugation at 800 g for 5 min at room temperature. Cells were resuspended in 1 mL S2+60 g/L glycerol and 10 g/L methyl palmitate was added. Cell were incubated for 96h at 1000 rpm, 28° C. FIG. 52 and FIG. 53 show fatty alcohol titers and fatty acid titers, respectively, for the 24 well plate screening experiments.

Extraction of cultivation samples. 0.5 mL of each sample were harvested directly into crimp vials. The crimp vials were centrifuged at 800 g for 5 min. The supernatant was removed, and the samples were sealed and stored at −20° C. until analysis. Samples were resuspended in 500 μL methanol containing 5% KOH and incubated for 60 min at 65° C. to 85° C. 200 μL 5N HCL was added to each crimp vial after cool down step. 600 μL chloroform containing 1 mM C17Me were added and sealed again. Samples were mixed and centrifuged. 500 μL of the chloroform was transferred to a new GC vial and dried to completeness at 85° C. for 30 min. Samples were resuspend in 100 μL BSTFA and incubated at room temperature for 1 h. Samples were analyzed according to standard protocol using gas chromatography.

TABLE 24

New FAR enzyme library

Organism
Enzyme ID
Gen ID

Agrotis
segetum

FAR027
AID66655.1

Euglena
gracilis

FAR028
GU733919

Ostrinia
palustralis

FAR029
MIQSS3

Ostrinia
latipennis

FAR030
MIRG73

Ostrinia
nubilalis

FAR031
FJ807735

Ostrinia
nubilalis

FAR032
FJ807736

Ostrinia
zealis

FAR033
MIQSS9

Yponomeuta
evonymellus

FAR034
GQ907232

Yponomeuta
padellus

FAR035
GQ907235

Yponomeuta
rorrellus

FAR036
GQ907234

Tyto
alba

FAR037
JN638549

Ostrinia
scapulalis

FAR038
EU817405

Spodoptera
exigua

FAR039
KR781121.1

Spodoptera
littoralis

FAR040
KR781120

H.
virescens

FAR041
EZ407233

H.
subflexa

FAR042
AFD04726.1

H.
amigera

FAR043
AKD01773.1

H.
assaulta

FAR044
AFD04727.1

Additional FAR variants and additional bacterial FAR enzymes are listed in Table 25 and Table 26, respectively. These FAR enzymes are tested as described above for fatty alcohol and fatty acid production.

TABLE 25

New FAR Enzyme library with phosphorylation point mutants

Amino acid mutations
Enzyme ID

HaFAR S12A
pFAR055

SlFAR S10A
pFAR056

SlFAR S13A
pFAR057

SlFAR S61A
pFAR058

SlFAR S104A
pFAR059

SlFAR S310A
pFAR060

SlFAR S314A
pFAR061

SlFAR S419A
pFAR062

TABLE 26

Additional bacterial FAR enzyme library

Organism
Gen ID

Spongiibacter tropicus

WP_022959477.1

Oleiphilus sp. HI0086
KZY30886.1

Marinobacter lipolyticus

WP_012138772.1

Marinobacter antarcticus

WP_072799080.1

SEQUENCE LISTING

SEQ ID NO: 1

Agrotis segetum FAR_S. cerevisiae codon opt

ATGCCAGTTTTGACTTCTAGAGAAGATGAAAAGTTGTCAGTTCCAGAATTTTACGCTGGTA

AATCTATCTTCGTTACAGGTGGTACTGGTTTCTTGGGTAAAGTTTTTATTGAAAAGTTGTT

GTACTGTTGTCCAGATATTGATAAAATCTATATGTTAATTAGAGAAAAGAAAAATTTGTCT

ATTGATGAAAGAATGTCAAAGTTCTTGGATGATCCATTATTTTCTAGATTGAAGGAAGAAA

GACCTGGTGACTTGGAAAAGATTGTTTTGATTCCAGGTGACATTACAGCTCCAAATTTGGG

TTTATCAGCAGAAAACGAAAGAATTTTGTTAGAAAAAGTTTCTGTTATTATTAATTCAGCT

GCAACTGTTAAGTTTAATGAACCATTGCCAATCGCTTGGAAGATTAATGTTGAAGGTACAA

GAATGTTGTTGGCATTGTCTAGAAGAATGAAGAGAATCGAAGTTTTTATTCATATTTCTAC

TGCTTACTCAAATGCATCTTCAGATAGAATCGTTGTTGATGAAATCTTGTATCCAGCTCCA

GCAGATATGGATCAAGTTTACCAATTGGTTAAAGATGGTGTTACAGAAGAAGAAACTGAAA

GATTGTTGAACGGTTTGCCAAACACTTACACTTTTACTAAGGCTTTGACAGAACATTTGGT

TGCAGAACATCAAACATACGTTCCAACTATCATCATCAGACCATCTGTTGTTGCTTCAATT

AAAGATGAACCAATCAGAGGTTGGTTATGTAATTGGTTTGGTGCTACAGGTATCTCTGTTT

TTACTGGAAAGGGTTTGAACAGAGTTTTGTTGGGTAAAGCTTCAAACATCGTTGATGTTAT

CCCAGTTGATTACGTTGCAAATTTGGTTATTGTTGCTGGTGCAAAATCTGGTGGTGAAAAA

TCAGATGAATTAAAGATCTATAACTGTTGTTCTTCAGATTGTAACCCAGTTACTTTGAAGA

AAATTATTAAAGAGTTTAGTGAAGATAGTATTAAAAATAAGTCTCATATTATGCCATTGCC

AGGTTGGTTCGTTTTTACTAAGTACAAGTGGTTGTTGACATTGTTAACTATTATTTTTCAA

ATGTTACCAATGTATTTGGCTGATGTTTACAGAGTTTTGACAGGTAAAATCCCAAGATACA

TGAAGTTGCATCATTTGGTTATTCAAACAAGATTGGGTATCGATTTCTTTACTTCTCATTC

ATGGGTTATGAAGACAGATAGAGTTAGAGAATTATTCGGTTCTTTGTCATTGGGAGAAAAG

CATATGTTTCCATGTGATCCATCTTCAATCGATTGGACAGATTATTTGCAATCATACTGTT

ACGGTGTTAGAAGATTTTTGGAAAAGAAGAAATAA

SEQ ID NO: 2

Spodoptera littoralis FAR1_S. cerevisiae codon opt

ATGGTTGTTTTGACTTCAAAGGAAAAATCAAACATGTCTGTTGCTGATTTCTACGCTGGTA

AATCTGTTTTTATTACAGGTGGTACTGGTTTCTTGGGTAAAGTTTTTATTGAAAAGTTGTT

GTACTCATGTCCAGATATTGATAAAATCTATATGTTGATCAGAGAAAAGAAAGGTCAATCT

ATCAGAGAAAGATTAACTAAAATTGTTGATGATCCATTGTTTAATAGATTGAAGGATAAGA

GACCAGATGATTTGGGTAAAATCGTTTTGATCCCAGGTGACATCACAGTTCCAGGTTTGGG

TATTTCTGAAGAAAACGAAACAATCTTGACTGAAAAAGTTTCAGTTGTTATTCATTCTGCT

GCAACTGTTAAGTTTAATGAACCATTGGCTACTGCATGGAACGTTAACGTTGAAGGTACAA

GAATGATCATGGCATTATCAAGAAGAATGAAGAGAATCGAAGTTTTTATTCATATTTCTAC

TGCTTACACTAACACAAACAGAGGAGTTATTGATGAAGTTTTGTATCCACCACCAGCTGAT

ATCAACGATGTTCATCAACATGTTAAAAATGGTGTTACAGAAGAAGAAACTGAAAAGATTT

TGAACGGTAGACCAAACACTTACACTTTTACTAAGGCTTTGACTGAACATTTGGTTGCAGA

AAACCAATCATACATGCCAACAATCATTGTTAGACCATCTATTGTTGGTGCTATTAAAGAT

GATCCAATTAGAGGTTGGTTGGCTAATTGGTATGGTGCAACAGGTTTGTCAGTTTTTACTG

CAAAGGGTTTGAACAGAGTTATATATGGTCATTCTAACCATGTTGTTGATTTGATTCCAGT

TGATTACGTTGCTAATTTGGTTATTGTTGCTGGTGCAAAGACATACCATTCAAACGAAGTT

ACTATCTATAACTCTTGTTCTTCATCTTGTAACCCAATCACTATGAAGAGATTGGTTGGTT

TGTTTATTGATTACACAGTTAAGCATAAGTCATACGTTATGCCATTGCCAGGTTGGTATGT

TTACTCTAACTACAAGTGGTTGGTTTTCTTGGTTACTGTTATTTTCCAAGTTATTCCAGCT

TACTTAGGTGACATTGGTAGAAGATTGTTAGGTAAAAATCCAAGATACTACAAGTTGCAAA

ATTTGGTTGCTCAAACACAAGAAGCAGTTCATTTCTTTACATCACATACTTGGGAAATTAA

ATCAAAGAGAACTTCTGAATTGTTTTCATCTTTGTCTTTGACAGATCAAAGAATGTTTCCA

TGTGATGCTAACAGAATCGATTGGACAGATTACATCACTGATTAGTGTTCTGGTGTTAGAC

AATTTTTGGAAAAGATTAAATAA

SEQ ID NO: 3

Helicoverpa armigera FAR3_S. cerevisiae codon opt

ATGGTTGTTTTGACTTCAAAGGAAACAAAGCCATCTGTTGCTGAATTTTACGCTGGTAAAT

CAGTTTTTATTACAGGTGTACTGGTTTCTTGGGTAAAGTTTTTATTGAAAAGTTGTTGTAC

TCTTGTCCAGATATTGAAAATATCTATATGTTGATCAGAGAAAAGAAAGGTTTGTCAGTTT

CTGAAAGAATTAAACAATTTTTAGATGATCCATTGTTTACAAGATTGAAGGATAAGAGACC

AGCTGATTTGGAAAAGATTGTTTTGATCCCAGGTGACATCACTGCACCAGATTTGGGTATT

AATTCTGAAAACGAAAAGATGTTGATTGAAAAAGTTTCAGTTATTATTCATTCTGCTGCAA

CTGTTAAGTTTAATGAACCATTACCAACAGCTTGGAAGATTAATGTTGAAGGTACTAGAAT

GATGTTGGCATTGTCAAGAAGAATGAAGAGAATCGAAGTTTTTATTCATATTTCTACAGCT

TACACTAACACAAACAGAGAAGTTGTTGATGAAATCTTGTATCCAGCTCCAGCAGATATCG

ATCAAGTTCATCAATACGTTAAGGATGGTATCTCAGAAGAAGATACTGAAAAGATTTTGAA

CGGTAGACCAAACACTTACACTTTTACTAAGGCTTTGACAGAACATTTGGTTGCTGAAAAT

CAAGCATACGTTCCAACTATTATTGTTAGACCATCTGTTGTTGCTGCAATTAAAGATGAAC

CATTGAAAGGTTGGTTGGGTAATTGGTTTGGTGCTACAGGTTTGACTGTTTTTACAGCAAA

GGGTTTGAACAGAGTTATATATGGTCATTCTTCATACATCGTTGATTTGATCCCAGTTGAT

TACGTTGCTAATTTGGTTATTGCTGCAGGTGCAAAATCTTCAAAGTCAACAGAATTGAAGG

TTTACAACTGTTGTTCTTCATCTTGTAACCCAGTTACTATCGGTACATTGATGTCAATGTT

CGCTGATGATGCAATTAAACAAAAATCTTACGCTATGCCATTGCCAGGTTGGTACATTTTT

ACAAAGTACAAGTGGTTGGTTTTGTTGTTGACATTTTTGTTCCAAGTTATTCCAGCATACG

TTACTGATTTGTCAAGACATTTGATCGGTAAATCTCCAAGATACATCAAGTTGCAATCATT

GGTTAACCAAACTAGATCATCTATCGATTTCTTTACAAACCATTCTTGGGTTATGAAAGCT

GATAGAGTTAGAGAATTGTACGCTTCATTGTCTCCAGCTGATAAGTACTTATTCCCATGTG

ATCCAACTGATATCAACTGGACACATTACATCCAAGATTACTGTTGGGGTGTTAGACATTT

CTTGGAAAAGAAATCTTACGAATAA

SEQ ID NO: 4
pOLE1 cassette

CTTGCTGAAAAGATGATGTTCTGAGGTATTCGTATCGCTAGCTTGATACGCTTTTAACAAA

AGTAAGCTTTTCGTTTGCAGGTTTGGTTACTTTTCTGTACGAGATGATATCGCTAAGTTTA

TAGTCATCTGTGAAATTTCTCAAAAACCTCATGGTTTCTCCATCACCCATTTTTCATTTCA

TTTGCCGGGCGGAAAAAAAAAAGGAAAAAAAAAAAAAAAAAAAATAAATGACACATGGAAA

TAAGTCAAGGATTAGCGGATATGTAGTTCCAGTCCGGGTTATACCATCACGTGATAATAAA

TCCAAATGAGAATGAGGGTGTCATATCTAATCATTATGCACGTCAAGATTCTCCGTGACTA

TGGCTCTTTTCTGAAGCATTTTTCGGGCGCCCGGTGGCCAAAAACTAACTCCGAGCCCGGG

CATGTCCCGGGGTTAGCGGGCGCAACAAAGGCGCTTATCTGGTGGGCTTCCGTAGAAGAAA

AAAAGCTGTTGAGCGAGCTATTTCGGGTATCCCAGCCTTCTCTGCAGACCGCCCCAGTTGG

CTTGGCTCTGGTGCTGTTCGTTAGCATCACATCGCCTGTGACAGGCAGAGGTAATAACGGC

TTAAGGTTCTCTTCGCATAGTCGGCAGCTTTCTTTCGGACGTTGAACACTCAACAAACCTT

ATCTAGTGCCCAACCAGGTGTGCTTCTACGAGTCTTGCTCACTCAGACACACCTATCCCTA

TTGTTACGGCTATGGGGATGGCACACAAAGGTGGAAATAATAGTAGTTAACAATATATGCA

GCAAATCATCGGCTCCTGGCTCATCGAGTCTTGCAAATCAGCATATACATATATATATGGG

GGCAGATCTTGATTCATTTATTGTTCTATTTCCATCTTTCCTAGTTCTGTTTCCGTTTATA

TTTTGTATTACGTAGAATAGAACATCATAGTAATAGATAGTTGTGGTGATCATATTATAAA

CAGCACTAAAACATTACAACAAAGAATGCCAACTTCTGGAACTACTATTGAATTGATTGAC

GACCAATTTCCAAAGGATGACTCTGCCAGCAGTGGCATTGTCGACACTAGTGCGGCCGCTC

ACATATGAAAGTATATACCCGCTTTTGTACACTATGTAGCTATAATTCAATCGTATTATTG

TAGCTCCGCACGACCATGCCTTAGAAATATCCGCAGCGCG

SEQ ID NO: 5
Extended OLE1 promoter region

CTTGCTGAAAAGATGATGTTCTGAGGTATTCGTATCGCTAGCTTGATACGCTTTTAACAAA

AGTAAGCTTTTCGTTTGCAGGTTTGGTTACTTTTCTGTACGAGATGATATCGCTAAGTTTA

TAGTCATCTGTGAAATTTCTCAAAAACCTCATGGTTTCTCCATCACCCATTTTTCATTTCA

TTTGCCGGGCGGAAAAAAAAAAGGAAAAAAAAAAAAAAAAAAAATAAATGACACATGGAAA

TAAGTCAAGGATTAGCGGATATGTAGTTCCAGTCCGGGTTATACCATCACGTGATAATAAA

TCCAAATGAGAATGAGGGTGTCATATCTAATCATTATGCACGTCAAGATTCTCCGTGACTA

TGGCTCTTTTCTGAAGCATTTTTCGGGCGCCCGGTGGCCAAAAACTAACTCCGAGCCCGGG

CATGTCCCGGGGTTAGCGGGCCCAACAAAGGCGCTTATCTGGTGGGCTTCCGTAGAAGAAA

AAAAGCTGTTGAGCGAGCTATTTCGGGTATCCCAGCCTTCTCTGCAGACCGCCCCAGTTGG

CTTGGCTCTGGTGCTGTTCGTTAGGATCACATCGCCTGTGACAGGCAGAGGTAATAACGGC

TTAAGGTTCTCTTCGCATAGTCGGCAGCTTTCTTTCGGACGTTGA

SEQ ID NO: 6
OLE1 promoter region

ACACTCAACAAACCTTATCTAGTGCCCAACCAGGTGTGCTTCTACGAGTCTTGCTCACTCA

GACACACCTATCCCTATTGTTACGGCTATGGGGATGGCACACAAAGGTGGAAATAATAGTA

GTTAACAATATATGCAGCAAATCATCGGCTCCTGGCTCATCGAGTCTTGCAAATCAGCATA

TACATATATATATGGGGGCAGATCTTGATTCATTTATTGTTCTATTTCCATCTTTCCTACT

TCTGTTTCCGTTTATATTTTGTATTACGTAGAATAGAACATCATAGTAATAGATAGTTGTG

GTGATCATATTATAAACAGCACTAAAACATTACAACAAAGA

SEQ ID NO: 7
OLE1 27 aa leader

ATGCCAACTTCTGGAACTACTATTGAATTGATTGACGACCAATTTCCAAAGGATGACTCTG

CCAGCAGTGGCATTGTCGAC

SEQ ID NO: 8
Vsp13 terminator region

TCACATATGAAAGTATATACCCGCTTTTGTACACTATGTAGCTATAATTCAATCGTATTAT

TGTAGCTCCGCACGACCATGCCTTAGAAATATCCGCAGCGCG

SEQ ID NO: 9

T. ni desaturase

ATGGCTGTGATGGCTCAAACAGTACAAGAAACGGCTACAGTGTTGGAAGAGGAAGCTCGCA

CAGTGACTCTTGTGGCTCCAAAGACAACGCCAAGGAAATATAAATATATATACACCAACTT

TCTTACATTTTCATATGCGCATTTAGCTGCATTATACGGACTTTATTTGTGCTTCACCTCT

GCGAAATGGGAAACATTGCTATTCTCTTTCGTACTCTTCCACATGTCAAATATAGGCATCA

CCGCAGGGGCTCACCGACTCTGGACTCACAAGACTTTCAAAGCCAAATTGCCTTTGGAAAT

TGTCCTCATGATATTCAACTCTTTAGCCTTTCAAAACACGGCTATTAGATGGGCTAGAGAA

CATCGGCTACATCACAAATACAGCGATACTGATGCTGATCCCCACAATGCGTCAAGAGGGT

TCTTCTACTCGCATGTTGGCTGGCTATTAGTAAAAAAACATCCCGATGTCCTGAAATATGG

AAAAACTATAGACATGTCGGATGTATACAATAATCCTGTGTTAAAATTTCAGAAAAAGTAC

GCAGTACCCTTAATTGGAACAGTTTGTTTTGCTCTGCCAACTTTGATTCCAGTCTACTGTT

GGGGCGAATCGTGGAACAACGCTTGGCACATAGCCTTATTTCGATACATATTCAATCTTAA

CGTGACTTTCCTAGTCAACAGTGCTGCGCATATCTGGGGGAATAAGCCTTATGATAAAAGC

ATCTTGCCCGCTCAAAACCTGCTGGTTTCCTTCCTAGCAAGTGGAGAAGGCTTCCATAATT

ACCATCACGTCTTTCCATGGGATTACCGCACAGCAGAATTAGGGAATAACTTCCTGAATTT

GACGACGCTGTTCATTGATTTTTGTGCCTGGTTTGGATGGGCTTATGACTTGAAGTCTGTA

TCAGAGGATATTATAAAACAGAGAGCTAAACGAACAGGTGACGGTTCTTCAGGGGTCATTT

GGGGATGGGACGACAAAGACATGGACCGCGATATAAAATCTAAAGCTAACATTTTTTATGC

TAAAAAGGAATGA

SEQ ID NO: 10

A. segetum desaturase

ATGGCTCAAGGTGTCCAAACAACTACGATATTGAGGGAGGAGGAGCCGTCATTGACTTTCG

TGGTACCTCAAGAACCGAGAAAGTATCAAATCGTGTACCCAAACCTTATCACATTTGGGTA

CTGGCATATAGCTGGTTTATACGGGCTATATTTGTGCTTTACTTCGGCAAAATGGCAAACA

ATTTTATTCAGTTTCATGCTCGTTGTGTTAGCAGAGTTGGGAATAACAGCCGGCGCTCACA

GGTTATGGGCCCACAAAACATATAAAGCGAAGCTTCCCTTACAAATTATCCTGATGATACT

GAACTCCATTGCCTTCCAAAATTCCGCCATTGATTGGGTGAGGGACCACCGTCTCCATCAT

AAGTACAGTGACACTGATGCAGACCCTCACAATGCTACTCGTGGTTTCTTCTATTCTCATG

TTGGATGGTTGCTCGTAAGAAAACATCCAGAAGTCAAGAGACGTGGAAAGGAACTTGACAT

GTCTGATATTTACAACAATCCAGTGCTGAGATTTCAAAAGAAGTATGCTATACCCTTCATC

GGGGCAATGTGCTTCGGATTACCAACTTTTATCCCTGTTTACTTCTGGGGAGAAACCTGGA

GTAATGCTTGGCATATCACCATGCTTCGGTACATCCTCAACCTAAACATTACTTTCCTGGT

CAACAGTGCTGCTCATATCTGGGGATACAAACCTTATGACATCAAAATATTGCCTGCCCAA

AATATAGCAGTTTCCATAGTAACCGGCGGCGAAGTTTCCATAACTACCACCACGTTTTTTC

CTTGGGATTATCGTGCAGCAGAATTGGGGAACAATTATCTTAATTTGACGACTAAGTTCAT

AGATTTCTTCGCTTGGATCGGATGGGCTTACGATCTTAAGACGGTGTCCAGTGATGTTATA

AAAAGTAAGGCGGAAAGAACTGGTGATGGGACGAATCTTTGGGGTTTAGAAGACAAAGGTG

AAGAAGATTTTTTGAAAATCTGGAAAGAGAATTAA

SEQ ID NO: 11

T. pseudonana desaturase

ACTAGTATGGACTTTCTCTCCGGCGATCCTTTCCGGACACTCGTCCTTGCAGCACTTGTTG

TCATCGGATTTGCTGCGGCGTGGCAATGCTTCTACCCGCCGAGCATCGTCGGCAAGCCTCG

TACATTAAGCAATGGTAAACTCAATACCAGAATCCATGGCAAATTGTACGACCTCTCATCG

TTTCAGCATCCAGGAGGCCCCGTGGCTCTTTCTCTTGTTCAAGGTCGCGACGGAACAGCTC

TATTTGAGTCACACCATCCCTTCATACCTCGAAAGAATCTACTTCAGATCCTCTCCAAGTA

CGAGGTTCCGTCGACTGAAGACTCTGTTTCCTTCATCGCCACCCTAGACGAACTCAATGGT

GAATCTCCGTACGATTGGAAGGACATTGAAAATGATGATTTCGTATCTGACCTACGAGCTC

TCGTAATTGAGCACTTTTCTCCTCTCGCCAAGGAAAGGGGAGTTTCACTCGTTGAGTCGTC

GAAGGCAACACCTCAGCGGTGGATGGTGGTTCTACTGCTCCTTGCGTCGTTCTTCCTGAGC

ATCCCATTATATTTGAGTGGTTCGTGGACTTTCGTTGTCGTCACTCCCATCCTCGCTTGGC

TGGCGGTTGTCAATTACTGGCACGATGCTACTCACTTTGCATTGAGCAGCAACTGGATTTT

GAATGCTGCGCTCCCATATCTCCTCCCTCTCCTATCGAGTCCGTCAATGTGGTATCATCAT

CACGTCATTGGACATCACGCATACACCAACATTTCCAAAAGAGATCCAGATCTTGCTCACG

CTCCACAACTCATGAGAGAACACAAGAGTATCAAATGGAGACCATCTCACTTAAATCAAAC

ACAGCTTCCGCGGATTCTCTTCATCTGGTCGATTGCAGTCGGTATTGGGTTGAACTTACTG

AACGACGTGAGAGCACTAACCAAGCTTTCATACAACAACGTTGTTCGGGTGGAGAAGATGT

CATCGTCGCGAACATTACTCCATTTCCTTGGACGTATGTTGCACATCTTTGTGACTACACT

TTGGCCCTTTTTGGCGTTTCCGGTGTGGAAGGCCATCGTTTGGGCGACTGTACCGAATGCC

ATACTGAGTTTGTGCTTCATGCTGAATACGCAAATCAATCACCTCATCAACACGTGTGCAC

ATGCTTCCGATAACAACTTTTACAAGCATCAAGTTGTAACTGCTCAGAACTTTGGCCGATC

AAGTGCCTTTTGCTTCATCTTCTCGGGAGGTCTCAACTACCAAATTGAACATCATTTGTTG

CCGACGGTGAACCATTGCCATTTGCCAGCTTTGGCCCCGGGTGTAGAGCGTTTGTGTAAGA

AACACGGGGTGACATACAACTCTGTTGAAGGATACAGAGAGGCCATCATTGCACACTTTGC

ACATACCAAAGATATGTCGACGAAGCCTACTGATTGA

SEQ ID NO: 12

A. transitella desaturase

ATGGTCCCTAACAAGGGTTCCAGTGACGTTTTGTCTGAACATTCTGAGCCCCAGTTCACTA

AACTCATAGCTCCACAAGCAGGGCCGAGGAAATACAAGATAGTGTATCGAAATTTGCTCAC

ATTCGGCTATTGGCACTTATCAGCTGTTTATGGGCTCTACTTGTGCTTTACTTGTGCGAAA

TGGGCTACCATCTTATTTGCATTTTTCTTATACGTGATCGCGGAAATCGGTATAACAGGTG

GCGCTCATAGGCTATGGGCACATCGGACTTATAAAGCCAAGTTGCCTTTAGAGATTTTGTT

ACTCATAATGAACTCTATTGCCTTCCAAGACACTGCTTTCACCTGGGCTCGTGATCACCGC

CTTCATCACAAATATTCGGATACTGACGCTGATCCCCACAATGCTACCAGAGGGTTTTTCT

ATTCACATGTAGGCTGGCTTTTGGTGAAGAAACACCCTGAAGTCAAAGCAAGAGGAAAATA

CTTGTCGTTAGATGATCTTAAGAATAATCCATTGCTTAAATTCCAAAAGAAATACGCTATT

CTAGTTATAGGCACGTTATGCTTCCTTATGCCAACATTTGTGCCCGTATACTTCTGGGGCG

AGGGCATCAGCACGGCCTGGAACATCAATCTATTGCGATACGTCATGAATCTTAACATGAC

TTTCTTAGTTAACAGTGCAGCGCATATCTTTGGCAACAAACCATACGATAAGAGCATAGCC

TCAGTCCAAAATATTTCAGTTAGCTTAGCTACTTTTGGCGAAGGATTCCATAATTACCATC

ACACTTACCCCTGGGATTATCGTGCGGCAGAATTAGGAAATAATAGGCTAAATATGACTAC

TGCTTTCATAGATTTCTTCGCTTGGATCGGCTGGGCTTATGACTTGAAGTCTGTGCCACAA

GAGGCCATTGCAAAAAGGTGTGCGAAAACTGGCGATGGAACGGATATGTGGGGTCGAAAAA

GATAA

SEQ ID NO: 13

H. zea desaturase

ATGGCCCAAAGCTATCAATCAACTACGGTTTTGAGTGAGGAGAAAGAACTAACACTGCAAC

ATTTGGTGCCCCAAGCATCGCCCAGGAAGTATCAAATAGTGTATCCGAACCTCATTACGTT

TGGTTACTGGCACATAGCCGGACTTTATGGCCTTTACTTGTGCTTCACTTCTGCTAAATGG

GCTACGATTTTATTCAGCTACATCCTCTTCGTGTTAGCAGAAATAGGAATCACGGCTGGCG

CTCACAGACTCTGGGCCCACAAAACTTACAAAGCGAAACTACCATTAGAAATACTCTTAAT

GGTATTCAACTCCATCGCTTTTCAAAACTCAGCCATTGACTGGGTGAGGGACCACCGACTC

CACCATAAGTATAGCGATACAGATGCTGATCCCCACAATGCCAGCCGAGGGTTCTTTTATT

CCCATGTAGGATGGCTACTTGTGAGAAAACATCCTGAAGTCAAAAAGCGAGGGAAAGAACT

CAATATGTCCGATATTTACAACAATCCTGTCCTGCGGTTTCAGAAAAAATACGCCATACCC

TTCATTGGGGCTGTTTGTTTCGCCTTACCTACAATGATACCTGTTTACTTCTGGGGAGAAA

CCTGGTCCAATGCTTGGCATATCACCATGCTTCGCTACATCATGAACCTCAATGTCACCTT

TTTGGTAAACAGCGCTGCTCATATATGGGGAAACAAGCCTTATGACGCAAAAATATTACCT

GCACAAAATGTAGCTGTGTCGGTCGCCACTGGTGGAGAAGGTTTCCATAATTACCACCATG

TCTTCCCCTGGGATTATCGAGCAGCGGAACTCGGTAACAATAGCCTCAATCTGACGACTAA

ATTCATAGATTTATTCGCAGCAATCGGATGGGCATATGATCTGAAGACGGTTTCGGAGGAT

ATGATAAAACAAAGGATTAAACGCACTGGAGATGGAACGGATCTTTGGGGACACGAACAAA

ACTGTGATGAAGTGTGGGATGTAAAAGATAAATCAAGTTAA

SEQ ID NO: 14
mCherry C. tropicalis optimized

ATGGTTTCTAACGGTGAAGAAGACAACATGGCAATCATCAAGGAATTTATGCGTTTTAAGG

TCCATATGGAAGGCTCCGTTAACGGCCACGAGTTCGAGATCGAGGGAGAAGGTGAGGGTAG

ACCATACGAAGGTACTCAAACCGCCAAGTTGAAAGTTACAAAGGGTGGTCCATTGCCATTT

GCTTGGGATATCTTGTCCCCACAATTTATGTACGGATCAAAGGCATATGTCAAGCATCCTG

CCGACATCCCAGATTACTTGAAGTTATCCTTTCCAGAAGGTTTTAAGTGGGAGAGAGTTAT

GAACTTTGAAGATGGCGGAGTTGTTACTGTTACTCAGGACTCTTCCTTGCAAGATGGTGAA

TTTATCTATAAAGTGAAATTGAGAGGTACTAACTTTCCATCCGACGGTCCAGTCATGCAAA

AGAAGAGAATGGGTTGGGAGGCTTCTTCCGAAAGAATGTACCCAGAAGACGGTGCATTGAA

AGGTGAAATCAAGCAACGTTTAAAGTTGAAGGACGGTGGTCACTACGATGCCGAGGTCAAG

ACCACTTATAAGGCTAAGAAGCCAGTCCAATTGCCAGGTGCTTATAACGTTAACATCAAGT

TAGATATTACTTCACACAACGAAGACTACACAATCGTTGAACAATATGAAAGAGCCGAAGG

TAGACATTCTACCGGCGGCATGGACGAGTTATATAAGTAG

SEQ ID NO: 15
CaOLE1-A. segetum Z11 desaturase

ATGACTACAGTTGAACAACTTGAAACTGTTGATATCACTAAATTGAATGCCATTGCTGCTG

GTACTAATAAGAAGGTGCCAATGGCTCAAGGTGTCCAAACAACTACGATATTGAGGGAGGA

AGAGCCGTCATTGACTTTCGTGGTACCTCAAGAACCGAGAAAGTATCAAATCGTGTACCCA

AACCTTATCACATTTGGGTACTGGCATATAGCTGGTTTATACGGGCTATATTTGTGCTTTA

CTTCGGCAAAATGGCAAACAATTTTATTCAGTTTCATGCTCGTTGTGTTAGCAGAGTTGGG

AATAACAGCCGGCGCTCACAGGTTATGGGCCCACAAAACATATAAAGCGAAGCTTCCCTTA

CAAATTATCTTAATGATATTAAACTCCATTGCCTTCCAAAATTCCGCCATTGATTGGGTGA

GGGACCACCGTCTCCATCATAAGTACAGTGACACTGATGCAGACCCTCACAATGCTACTCG

TGGTTTCTTCTATTCTCATGTTGGATGGTTGCTCGTAAGAAAACATCCAGAAGTCAAGAGA

CGTGGAAAGGAACTTGACATGTCTGATATTTACAACAATCCAGTGTTAAGATTTCAAAAGA

AGTATGCTATACCCTTCATCGGGGCAATGTGCTTCGGATTACCAACTTTTATCCCTGTTTA

CTTCTGGGGAGAAACCTGGAGTAATGCTTGGCATATCACCATGCTTCGGTACATCCTCAAC

CTAAACATTACTTTCTTAGTCAACAGTGCTGCTCATATCTGGGGATACAAACCTTATGACA

TCAAAATATTGCCTGCCCAAAATATAGCAGTTTCCATAGTAACCGGCGGCGAACTTTCCAT

AACTACCACCACGTTTTTTCCTTGGGATTATCGTGCAGCAGAATTGGGGAACAATTATCTT

AATTTGACGACTAAGTTCATAGATTTCTTCGCTTGGATCGGATGGGCTTACGATCTTAAGA

CGGTGTCCAGTGATGTTATAAAAAGTAAGGCGGAAAGAACTGGTGATGGGACGAATCTTTG

GGGTTTAGAAGACAAAGGTGAAGAAGATTTTTTGAAAATCTGGAAAGACAATTAA

SEQ ID NO: 16

A. segetum Z11 desaturase

ATGGCTCAAGGTGTCCAAACAACTACGATATTGAGGGAGGAAGAGCCGTCATTGACTTTCG

TGGTACCTCAAGAACCGAGAAAGTATCAAATCGTGTACCCAAACCTTATCACATTTGGGTA

CTGGCATATAGCTGGTTTATACGGGCTATATTTGTGCTTTACTTCGGCAAAATGGCAAACA

ATTTTATTCAGTTTCATGCTCGTTGTGTTAGCAGAGTTGGGAATAACAGCCGGCGCTCACA

GGTTATGGGCCCACAAAACATATAAAGCGAAGCTTCCCTTACAAATTATCTTAATGATATT

AAACTCCATTGCCTTCCAAAATTCCGCCATTGATTGGGTGAGGGACCACCGTCTCCATCAT

AAGTACAGTGACACT

GATGCAGACCCTCACAATGCTACTCGTGGTTTCTTCTATTCTCATGTTGGATGGTTGCTCG

TAAGAAAACATCCAGAAGTCAAGAGACGTGGAAAGGAACTTGACATGTCTGATATTTACAA

CAATCCAGTGTTAAGATTTCAAAAGAAGTATGCTATACCCTTCATCGGGGCAATGTGCTTC

GGATTACCAACTTTTATCCCTGTTTACTTCTGGGGAGAAACCTGGAGTAATGCTTGGCATA

TCACCATGCTTCGGTACATCCTCAACCTAAACATTACTTTCTTAGTCAACAGTGCTGCTCA

TATCTGGGGATACAAACCTTATGACATCAAAATATTGCCTGCCCAAAATATAGCAGTTTCC

ATAGTAACCGGCGGCGAAGTTTCCATAACTACCACCACGTTTTTTCCTTGGGATTATCGTG

CAGCAGAATTGGGGAACAATTATCTTAATTTGACGACTAAGTTCATAGATTTCTTCGCTTG

GATCGGATGGGCTTACGATCTTAAGACGGTGTCCAGTGATGTTATAAAAAGTAAGGCGGAA

AGAACTGGTGATGGGACGAATCTTTGGGGTTTAGAAGACAAAGGTGAAGAAGATTTTTTGA

AAATCTGGAAAGACAATTAA

SEQ ID NO: 17

A. transitella Z11 desaturase

ATGGTCCCTAACAAGGGTTCCAGTGACGTTTTGTCTGAACATTCTGAGCCCCAGTTCACTA

AACTCATAGCTCCACAAGCAGGGCCGAGGAAATACAAGATAGTGTATCGAAATTTGCTCAC

ATTCGGCTATTGGCACTTATCAGCTGTTTATGGGCTCTACTTGTGCTTTACTTGTGCGAAA

TGGGCTAGCATCTTATTTGCATTTTTCTTATACGTGATCGCGGAAATCGGTATAACAGGTG

GCGCTCATAGGCTATGGGCACATCGGACTTATAAAGCCAAGTTGCCTTTAGAGATTTTGTT

ACTCATAATGAATTCTATTGCCTTCCAAGACACTGCTTTCACCTGGGCTCGAGATCACCGC

CTTCATCACAAATATTCGGATACTGACGCTGATCCCCACAATGCTACCAGAGGGTTTTTCT

ATTCACATGTAGGCTGGCTTTTGGTGAAGAAACACCCTGAAGTCAAAGCAAGAGGAAAATA

CTTGTCGTTAGATGATCTTAAGAATAATCCATTGCTTAAATTCCAAAAGAAATACGCTATT

CTAGTTATAGGCACGTTATGCTTCCTTATGCCAACATTTGTGCCCGTATACTTCTGGGGCG

AGGGCATCAGCACGGCCTGGAACATCAATCTATTGCGATACGTCATGAATCTTAACATGAC

TTTCTTAGTTAACAGTGCAGCGCATATCTTTGGCAACAAACCATACGATAAGAGCATAGCC

TCAGTCCAAAATATTTCAGTTAGCTTAGCTACTTTTGGCGAAGGATTCCATTATTACCATC

ACACTTACCCCTGGGATTATCGTGCGGCAGAATTAGGAAATAATAGGCTAAATATGACTAG

TGCTTTCATAGATTTCTTCGCTTGGATCGGCTGGGCTTATGACTTGAAGTCTGTGCCACAA

GAGGCCATTGCAAAAAGGTGTGCGAAAACTGGCGATGGAACGGATATGTGGGGTCGAAAAA

GATAA

SEQ ID NO: 18

T. ni Z11 desaturase

ATGGCTGTGATGGCTCAAACAGTACAAGAAACGGCTACAGTGTTGGAAGAGGAAGCTCGCA

CAGTGACTCTTGTGGCTCCAAAGACAACGCCAAGGAAATATAAATATATATACACCAACTT

TCTTACATTTTCATATGCGCATTTAGCTGCATTATACGGACTTTATTTGTGCTTCACCTCT

GCGAAATGGGAAACATTGCTATTCTCTTTCGTACTCTTCCACATGTCAAATATAGGCATCA

CCGCAGGGGCTCACCGACTCTGGACTCACAAGACTTTCAAAGCCAAATTGCCTTTGGAAAT

TGTCCTCATGATATTCAACTCTTTAGCCTTTCAAAACACGGCTATTACATGGGCTAGAGAA

CATCGGCTACATCACAAATACAGCGATACTGATGCTGATCCCCACAATGCGTCAAGAGGGT

TCTTCTACTCGCATGTTGGCTGGCTATTAGTAAAAAAACATCCCGATGTCTTAAAATATGG

AAAAACTATAGACATGTCGGATGTATACAATAATCCTGTGTTAAAATTTCAGAAAAAGTAC

GCAGTACCCTTAATTGGAACAGTTTGTTTTGCTCTTCCAACTTTGATTCCAGTCTACTGTT

GGGGCGAATCGTGGAACAACGCTTGGCACATAGCCTTATTTCGATACATATTCAATCTTAA

CGTGACTTTCCTAGTCAACAGTGCTGCGCATATCTGGGGGAATAAGCCTTATGATAAAAGC

ATCTTGCCCGCTCAAAACTTATTAGTTTCCTTCCTAGCAAGTGGAGAAGGCTTCGATAATT

ACCATCACGTCTTTCCATGGGATTACCGCACAGCAGAATTAGGGAATAACTTCTTAAATTT

GACGACGTTATTCATTGATTTTTGTGCCTGGTTTGGATGGGCTTATGACTTGAAGTCTGTA

TCAGAGGATATTATAAAACAGAGAGCTAAACCAACAGGTGACGGTTCTTCAGGGGTCATTT

GGGGATGGGACGACAAAGACATGGACCGCGATATAAAATCTAAAGCTAACATTTTTTATGC

TAAAAAGGAATGA

SEQ ID NO: 19

H. zea Z11 desaturase

ATGGCCCAAAGCTATCAATCAACTACGGTTTTGAGTGAGGAGAAAGAACTAACATTACAAC

ATTTGGTGCCCCAAGCATCGCCCAGGAAGTATCAAATAGTGTATCCGAACCTCATTACGTT

TGGTTACTGGCACATAGCCGGACTTTATGGCCTTTACTTGTGCTTCACTTCTGCTAAATGG

GCTAGGATTTTATTCAGCTACATCCTCTTCGTGTTAGCAGAAATAGGAATCACGGCTGGCG

CTCACAGACTCTGGGCCCACAAAACTTACAAAGCGAAACTAGCATTAGAAATACTCTTAAT

GGTATTCAACTCCATCGCTTTTCAAAACTCAGCCATTGACTGGGTGAGGGACCACCGACTC

CACCATAAGTATAGCGATACAGATGCTGATCCCCACAATGCCAGCCGAGGGTTCTTTTATT

CCCATGTAGGATGGCTACTTGTGAGAAAACATCCTGAAGTCAAAAAGCGAGGGAAAGAACT

CAATATGTCCGATATTTAGAACAATCCTGTCTTACGGTTTCAGAAAAAATACGCCATACCC

TTCATTGGGGCTGTTTGTTTCGCCTTACCTACAATGATACCTGTTTACTTCTGGGGAGAAA

CCTGGTCCAATGCTTGGCATATCACCATGCTTCGCTACATCATGAACCTCAATGTCACCTT

TTTGGTAAACAGCGCTGCTCATATATGGGGAAACAAGCCTTATGACGCAAAAATATTACCT

GCACAAAATGTAGCTGTGTCGGTCGCCACTGGTGGAGAAGGTTTCCATAATTACCACCATG

TCTTCCCCTGGGATTATCGAGCAGCGGAACTCGGTAACAATAGCCTCAATTTAACGACTAA

ATTCATAGATTTATTCGCAGCAATCGGATGGGCATATGATTTAAAGACGGTTTCGGAGGAT

ATGATAAAACAAAGGATTAAACGCACTGGAGATGGAACGGATCTTTGGGGACACGAACAAA

ACTGTGATGAAGTGTGGGATGTAAAAGATAAATCAAGTTAA

SEQ ID NO: 20

O. furnacalis Z9 desaturase

ATGGCTCCTAATATTAAGGACGGAGCTGATTTGAACGGAGTTTTATTTGAAGATGACGCTA

GCACCCCCGATTATGCCCTTGCCACGGCCCCAGTCCAGAAAGCAGACAACTATCCCAGAAA

ACTAGTGTGGAGAAACATCATACTCTTTGCATACCTTCACCTTGCCGCTGTGTATGGAGCA

TACCTATTCTTATTTTCAGCGAAATGGCAGACAGATATTTTTGCCTACATTCTTTACGTGA

TCTCAGGACTCGGCATCACAGCGGGAGCCCACCGCCTTTGGGCGCACAAGTCATACAAGGC

TAAGTGGCCACTTAGACTCATTCTTATTATCTTCAACACTGTATCATTCCAGGACTCTGCT

CTCGACTGGTCACGTGACCACCGCATGCACCACAAATACTCGGAGACCGACGCCGACCCGC

ACAACGCGACTCGAGGGTTCTTCTTCTCTCATATCGGCTGGTTATTAGTCCGCAAGCACCC

GGAATTAAAGAGAAAGGGCAAGGGATTAGACTTAAGCGACTTGTATGCTGATCCCATCCTC

CGTTTCCAGAAGAAGTACTATTTACTATTAATGCCTCTTGGCTGCTTCATCATGCCGACGG

TGGTCCCGGTGTACTTCTGGGGTGAGACTTGGACTAACGCTTTCTTCGTCGCCGCGCTCTT

CCGATACACCTTCATCCTCAATGTCACCTGGTTGGTCAACTCCGCCGCGCACAAGTGGGGC

CACAAGCCCTATGACAGCAGCATCAAGCCTTCCGAGAACCTCTCAGTCTCCTTATTCGCGT

TGGGCGAAGGATTCCACAACTACCACCACACATTCCCCTGGGACTACAAAACTGCCGAGCT

CGGCAACAACAGACTCAATTTCACAACAAACTTCATCAACTTCTTCGCTAAAATCGGATGG

GCTTACGACTTGAAAACGGTCTCCGACGAGATTATTCAGAATAGAGTCAAGCGCACAGGAC

ATGGCTCCCACCACTTATGGGGTTGGGGCGACAAGGATCAACCTAAAGAGGAGGTAAACGC

AGCCATTAGAATTAATCCTAAAGACGAGTAA

SEQ ID NO: 21

L. capitella Z9 desaturase

ATGCCGCCGAACGTGACAGAGGCGAACGGAGTGTTATTTGAGAATGACGTGCAGACTCCTG

ACATGGGGCTACAAGTGGCCCCTGTGCAGAAGGCTGACGAGCGTAAGATCCAGCTCGTTTG

GAGGAACATCATCGCTTTTGCATGTCTTCATTTAGCAGCTGTGTATGGAGCTTATTTATTC

TTCACCTCGGCTATATGGCAGACAGACATATTTGCATACATCCTTTACGTTATGTCTGGAT

TAGGAATCACGGCGGGAGCGCACAGATTATGGGCTCATAAGTCATACAAGGCGAAGTGGCC

GTTAAGATTAATCCTCGTCGCATTCAACACTTTGGCATTCCAGGATTCGGCAATCGACTGG

GCGCGCGACCACCGCATGCACCACAAGTACTCGGAGACGGATGCGGACCCACATAACGCCA

CTCGCGGCTTCTTCTTTTCGCACATTGGTTGGTTACTCTGCCGAAAACACCCGGAGCTAAA

GCGCAAGGGCCAGGGCCTCGACTTAAGTGACCTCTACGCAGATCCTATTATTCGCTTCCAA

AAGAAGTACTACTTATTGTTAATGCCGTTAGCCTGCTTTGTTCTTCCCACCATAATTCCGG

TCTACCTCTGGGGCGAGTCCTGGAAAAACGCGTTCTTCGTAGCTGCAATGTTCCGTTACAC

GTTCATCCTCAACGTAACATGGCTCGTCAACTCCGCCGCCCACAAATGGGGAGGCAAGCCC

TATGATAAGAACATCCAGCCCGCTCAGAACATCTCTGTAGCTATCTTCGCATTAGGCGAGG

GCTTCCACAACTACCACCACACGTTCCCCTGGGACTAGAAGACCGCTGAATTAGGAAACAA

CAGGTTAAATTTCACAACTTCGTTTATCAATTTCTTCGCAAGCTTCGGATGGGCCTACGAC

TTAAAGACCGTGTCGGACGAGATTATACAACAGCGCGTTAAGAGGACGGGAGATGGGAGCC

ATCACTTACGGGGCTGGGGCGACCAGGACATACCGGCCGAAGAAGCTCAAGCTGCTTTACG

CATTAACCGTAAAGATGATTAG

SEQ ID NO: 22

H. zea Z9 desaturase

ATGGCTCCAAATATATCGGAGGATGTGAACGGGGTGCTCTTCGAGAGTGATGCAGCGACGC

CGGACTTAGCGTTATCCACGCCGCCTGTGCAGAAGGCTGACAACAGGCCCAAGCAATTAGT

GTGGAGGAACATACTATTATTCGCGTATCTTCACTTAGCGGCTCTTTACGGAGGTTATTTA

TTCCTCTTCTCAGCTAAATGGCAGACAGACATATTTGCCTACATCTTATATGTGATCTCCG

GGCTTGGTATCACGGCTGGAGCACATCGCTTATGGGCCCACAAGTCCTACAAAGCTAAATG

GCCTCTCCGAGTTATCTTAGTCATCTTTAACACAGTGGCATTCCAGGATGCCGCTATGGAC

TGGGCGCGCGACCACCGCATGCATCACAAGTACTCGGAAACCGATGCTGATCCTCATAATG

CGACCCGAGGATTCTTCTTCTCTCACATTGGCTGGTTACTTGTCAGGAAACATCCCGACCT

TAAGGAGAAGGGCAAGGGACTCGACATGAGCGACTTACTTGCTGACCCCATTCTCAGGTTC

CAGAAAAAATACTACTTAATCTTAATGCCCTTGGCTTGCTTCGTGATGCCTACCGTGATTC

CTGTGTACTTCTGGGGTGAAACCTGGACCAACGCATTCTTTGTGGCGGCCATGTTCCGCTA

CGCGTTCATCCTAAATGTGACGTGGCTCGTCAACTCTGCCGCTCACAAGTGGGGAGACAAG

CCCTACGACAAAAGCATTAAGCCTTCCGAAAACTTGTCGGTCGCCATGTTCGCTCTCGGAG

AAGGATTCCACAACTACCACCACACTTTCCCTTGGGACTACAAAACTGCTGAGTTAGGCAA

CAACAAACTCAACTTCACTACCACCTTTATTAACTTCTTCGCTAAAATTGGCTGGGCTTAC

GACTTAAAGACAGTGTCTGATGATATCGTCAAGAACAGGGTGAAGCGCACTGGTGACGGCT

CCCACCACTTATGGGGCTGGGGAGACGAAAATCAATCCAAAGAAGAAATTGATGCCGCTAT

CAGAATCAATCCTAAGGACGATTAA

SEQ ID NO: 23

T. pseudonana Z11 desaturase

ATGGACTTTCTCTCCGGCGATCCTTTCCGGACACTCGTCCTTGCAGCACTTGTTGTCATCG

GATTTGCTGCGGCGTGGCAATGCTTCTACCCGCCGAGCATCGTCGGCAAGCCTCGTACATT

AAGCAATGGTAAACTCAATACCAGAATCCATGGCAAATTGTACGACCTCTCATCGTTTCAG

CATCCAGGAGGCCCCGTGGCTCTTTCTCTTGTTCAAGGTCGCGACGGAACAGCTCTATTTG

AGTCACACCATCCCTTCATACCTCGAAAGAATCTACTTCAGATCCTCTCCAAGTACGAGGT

TCCGTCGACTGAAGACTCTGTTTCCTTCATCGCCACCCTAGACGAACTCAATGGTGAATCT

CCGTACGATTGGAAGGAGATTGAAAATGATGATTTCGTATCTGAGCTAGGAGCTCTCGTAA

TTGAGCACTTTTCTCCTCTCGCCAAGGAAAGGGGAGTTTCACTCGTTGAGTCGTCGAAGGC

AACACCTCAGCGGTGGATGGTGGTTCTATTACTCCTTGCGTCGTTCTTCCTCAGCATCCCA

TTATATTTGAGTGGTTCGTGGACTTTCGTTGTCGTCACTCCCATCCTCGCTTGGTTAGCGG

TTGTCAATTACTGGCACGATGCTACTCACTTTGCATTGAGCAGCAACTGGATTTTGAATGC

TGCGCTCCCATATCTCCTCCCTCTCCTATCGAGTCCGTCAATGTGGTATCATCATCACGTC

ATTGGACATCACGCATACACCAACATTTCCAAAAGAGATCCAGATCTTGCTCACGCTCCAC

AACTCATGAGAGAACACAAGAGTATCAAATGGAGACCATCTCACTTAAATCAAACACAGCT

TCCGCGGATTCTCTTCATCTGGTCGATTGCAGTCGGTATTGGGTTGAACTTATTAAACGAC

GTGAGAGCACTAACCAAGCTTTCATACAACAACGTTGTTCGGGTGGAGAAGATGTCATCGT

CGCGAACATTACTCCATTTCCTTGGACGTATGTTGCACATCTTTGTGACTACACTTTGGCC

CTTTTTGGCGTTTCCGGTGTGGAAGGCCATCGTTTGGGCGACTGTACCGAATGCCATATTA

AGTTTGTGCTTCATGTTAAATACGCAAATCAATCACCTCATCAACACGTGTGCACATGCTT

CCGATAACAACTTTTACAAGCATCAAGTTGTAACTGCTCAGAACTTTGGCCGATCAAGTGC

CTTTTGCTTCATCTTCTCGGGAGGTCTCAACTACCAAATTGAACATCATTTGTTGCCGACG

GTGAACCATTGCCATTTGCCAGCTTTGGCCCCGGGTGTAGAGCGTTTGTGTAAGAAACACG

GGGTGACATACAACTCTGTTGAAGGATACAGAGAGGCCATCATTGCACACTTTGCACATAC

CAAAGATATGTCGACGAAGCCTACTGATTGA

SEQ ID NO: 24
Native T. ni Z11 desaturase

ATGGCTGTGATGGCTCAAACAGTACAAGAAACGGCTACAGTGTTGGAAGAGGAAGCTCGCA

CAGTGACTCTTGTGGCTCCAAAGACAACGCCAAGGAAATATAAATATATATACACCAACTT

TCTTACATTTTCATATGCGCATTTAGCTGCATTATACGGACTTTATTTGTGCTTCACCTCT

GCGAAATGGGAAACATTGCTATTCTCTTTCGTACTCTTCCACATGTGAAATATAGGCATCA

CCGCAGGGGCTCACCGACTCTGGACTCACAAGACTTTCAAAGCCAAATTGCCTTTGGAAAT

TGTCCTCATGATATTCAACTCTTTAGCCTTTCAAAACACGGCTATTAGATGGGCTAGAGAA

CATCGGCTACATCACAAATACAGCGATACTGATGCTGATCCCCACAATGCGTCAAGAGGGT

TCTTCTACTCGCATGTTGGCTGGCTATTAGTAAAAAAACATCCCGATGTCCTGAAATATGG

AAAAACTATAGACATGTCGGATGTATACAATAATCCTGTGTTAAAATTTCAGAAAAAGTAC

GCAGTACCCTTAATTGGAACAGTTTGTTTTGCTCTGCCAACTTTGATTCCAGTCTACTGTT

GGGGCGAATCGTGGAACAACGCTTGGCACATAGCCTTATTTCGATACATATTCAATCTTAA

CGTGACTTTCCTAGTCAACAGTGCTGCGCATATCTGGGGGAATAAGCCTTATGATAAAAGC

ATCTTGCCGGCTCAAAACCTGCTGGTTTCCTTCCTAGCAAGTGGAGAAGGCTTCCATAATT

ACCATCACGTCTTTCCATGGGATTACCGCACAGCAGAATTAGGGAATAACTTCCTGAATTT

GACGACGCTGTTCATTGATTTTTGTGCCTGGTTTGGATGGGCTTATGACTTGAAGTCTGTA

TCAGAGGATATTATAAAACAGAGAGCTAAACGAACAGGTGACGGTTCTTCAGGGGTCATTT

GGGGATGGGACGACAAAGACATGGACCGCGATATAAAATCTAAAGCTAACATTTTTTATGC

TAAAAAGGAATGA

SEQ ID NO: 25

H. zea Z11 desaturase

ATGGCCCAAAGCTATCAATCAACTACGGTTTTGAGTGAGGAGAAAGAACTAACACTGCAAC

ATTTGGTGCCCCAAGCATCGCCCAGGAAGTATCAAATAGTGTATCCGAACCTCATTACGTT

TGGTTACTGGCACATAGCCGGACTTTATGGCCTTTACTTGTGCTTCACTTCTGCTAAATGG

GCTACGATTTTATTCAGCTACATCCTCTTCGTGTTAGCAGAAATAGGAATCACGGCTGGCG

CTCACAGACTCTGGGCCCACAAAACTTACAAAGCGAAACTACCATTAGAAATACTCTTAAT

GGTATTCAACTCCATCGCTTTTCAAAACTCAGCCATTGACTGGGTGAGGGACCACCGACTC

CACCATAAGTATAGCGATACAGATGCTGATCCCCACAATGCCAGCCGAGGGTTCTTTTATT

CCCATGTAGGATGGCTACTTGTGAGAAAACATCCTGAAGTCAAAAAGCGAGGGAAAGAACT

CAATATGTCCGATATTTAGAACAATCCTGTCCTGCGGTTTCAGAAAAAATACGCCATACCC

TTCATTGGGGCTGTTTGTTTCGCCTTACCTACAATGATACCTGTTTACTTCTGGGGAGAAA

CCTGGTCCAATGCTTGGCATATCACCATGCTTCGCTACATCATGAACCTCAATGTCACCTT

TTTGGTAAACAGCGCTGCTCATATATGGGGAAACAAGCCTTATGACGCAAAAATATTACCT

GCACAAAATGTAGCTGTGTCGGTCGCCACTGGTGGAGAAGGTTTCCATAATTACCACCATG

TCTTCCCCTGGGATTATCGAGCAGCGGAACTCGGTAACAATAGCCTCAATCTGACGACTAA

ATTCATAGATTTATTCGCAGCAATCGGATGGGCATATGATCTGAAGACGGTTTCGGAGGAT

ATGATAAAACAAAGGATTAAACGCACTGGAGATGGAACGGATCTTTGGGGACACGAACAAA

ACTGTGATGAAGTGTGGGATGTAAAAGATAAATCAAGTTAA

SEQ ID NO: 26

T. ni Z11 desaturase Homo sapiens optimized

ATGGCCGTGATGGCCCAGACCGTGCAGGAGACCGCAACAGTGCTGGAGGAGGAGGCAAGGA

CCGTGACACTGGTGGCACCCAAGACCACACCTAGAAAGTACAAGTATATCTACACCAACTT

CCTGACCTTCAGCTACGCACACCTGGCCCCCCTGTATGGACTCTACCTGTGCTTTACCTCC

GCCAAGTGGGAGACACTGCTGTTCTCTTTTGTGCTGTTCCACATGAGCAATATCGGAATCA

CCGCAGGAGCACACAGGCTGTGGACCCACAAGACATTCAAGGCCAAGCTGCCTCTGGAGAT

CGTGCTGATGATCTTCAACTCTCTGGCCTTTCAGAATACCGCCATCACATGGGCCCGGGAG

CACAGACTGCACCACAAGTATAGCGACACCGATGCAGACCCACACAACGCAAGCAGGGGCT

TCTTTTACTCCCACGTGGGCTGGCTGCTGGTGAAGAAGCACCCCGACGTGCTGAAGTATGG

CAAGACAATCGACATGTCCGACGTGTACAACAATCCCGTGCTGAAGTTTCAGAAGAAGTAT

GCCGTGCCTCTGATCGGCACCGTGTGCTTCGCCCTGCCAACACTGATCCCCGTGTATTGTT

GGGGCGAGTCTTGGAACAATGCCTGGCACATCGCCCTGTTCCGGTACATCTTTAACCTGAA

TGTGACCTTTCTGGTGAACTCCGCCGCCCACATCTGGGGCAATAAGCCTTACGACAAGTCT

ATCCTGCCAGCCCAGAACCTGCTGGTGTCCTTCCTGGCCTCTGGCGAGGGCTTTCACAATT

ATCACCACGTGTTCCCATGGGACTACAGGACCGCAGAGCTGGGCAACAATTTTCTGAACCT

GACCACACTGTTCATCGATTTTTGTGCCTGGTTCGGCTGGGCCTATGACCTGAAGTCTGTG

AGCGAGGATATCATCAAGCAGAGGGCAAAGAGGACAGGCGATGGCAGCTCCGGCGTGATCT

GGGGATGGGACGATAAGGATATGGACAGAGATATCAAGAGCAAGGCCAATATCTTCTACGC

CAAGAAGGAGTGA

SEQ ID NO: 27

H. zea Z11 desaturase Homo sapiens optimized

ATGGCACAGTCATATCAGAGCACTACCGTCCTGAGCGAAGAGAAGGAACTGACACTGCAGC

ACCTGGTCCCACAGGCATCACCTAGAAAGTACCAGATCGTGTATCCAAACCTGATCACCTT

CGGCTACTGGCACATCGCCGGCCTGTACGGCCTGTATCTGTGCTTTAGCTCCGCCAAGTGG

GCCACAATCCTGTTCTCTTACATCCTGTTTGTGCTGGCAGAGATCGGAATCACCGCAGGAG

CACACAGACTGTGGGCACACAAGACATATAAGGCCAAGCTGCCCCTGGAGATCCTGCTGAT

GGTGTTCAACAGCATCGCCTTTCAGAATTCCGCCATCGATTGGGTGCGGGACCACAGACTG

CACCACAAGTACTCCGACACCGATGCCGACCCCCACAACGCCTCTAGGGGCTTCTTTTATA

GCCACGTGGGATGGCTGCTGGTGCGGAAGCACCCTGAGGTGAAGAAGAGAGGCAAGGAGCT

GAATATGTCTGATATCTACAACAATCCTGTGCTGCGCTTCCAGAAGAAGTATGCCATCCCA

TTCATCGGCGCCGTGTGCTTTGCCCTGCCCACCATGATCCCCGTGTACTTTTGGGGCGAGA

CATGGAGCAACGCCTGGCACATCACAATGCTGCGGTATATCATGAACCTGAATGTGACATT

CCTGGTGAACTCCGCCGCCCACATCTGGGGCAATAAGCCATACGACGCCAAGATCCTGCCC

GCCCAGAACGTGGCCGTGAGCGTGGCAACCGGAGGAGAGGGCTTCCACAATTACCACCACG

TGTTTCCTTGGGATTATCGGGCCGCCGAGCTGGGCAACAATTCTCTGAATCTGACCACAAA

GTTCATCGACCTGTTTGCCGCCATCGGCTGGGCCTATGATCTGAAGACAGTGAGCGAGGAC

ATGATCAAGCAGAGGATCAAGCGCACCGGCGATGGCACAGACCTGTGGGGGCACGAGCAGA

ACTGTGATGAAGTGTGGGATGTGAAAGACAAGTCCTCCTAA

SEQ ID NO: 28

Y. lipolytica OLE1 leader - T. ni Z11 desaturase Homo sapiens

optimized

ATGGTGAAGAACGTGGACCAGGTGGATCTGTCTCAGGTGGACACCATCGCAAGCGGAAGGG

ATGTGAATTATAAGGTGAAGTACACATCTGGCGTGAAGACCACACCAAGAAAGTACAAGTA

TATCTACACCAACTTCCTGACATTTTCTTACGCCCACCTGGCCGCCCTGTATGGCCTGTAC

CTGTGCTTTACCAGCGCCAAGTGGGAGACACTGCTGTTCTCCTTTGTGCTGTTCCACATGT

CTAATATCGGAATCACCGCAGGAGCACACAGGCTGTGGACCCACAAGACATTCAAGGCCAA

GCTGCCCCTGGAGATCGTGCTGATGATCTTCAACTCCCTGGCCTTTCAGAATACCGCCATC

ACATGGGCCCGGGAGCACAGACTGCACCACAAGTATTCTGACACCGATGCAGACCCACACA

ACGCAAGCAGGGGCTTCTTTTACTCCCACGTGGGCTGGCTGCTGGTGAAGAAGCACCCTGA

CGTGCTGAAGTATGGCAAGACAATCGACATGAGCGACGTGTACAACAATCCTGTGCTGAAG

TTTCAGAAGAAGTATGCCGTGCCACTGATCGGCACCGTGTGCTTCGCCCTGCCCACACTGA

TCCCCGTGTACTGTTGGGGCGAGTCCTGGAACAATGCCTGGCACATCGCCCTGTTCCGGTA

CATCTTTAACCTGAATGTGACCTTTCTGGTGAACAGCGCCGCCCACATCTGGGGCAATAAG

CCATACGACAAGTCCATCCTGCCCGCCCAGAACCTGCTGGTGTCCTTCCTGGCCTCTGGCG

AGGGCTTTCACAATTATCACCACGTGTTCCCTTGGGACTACAGGACCGCAGAGCTGGGCAA

CAATTTTCTGAACCTGACCACACTGTTCATCGATTTTTGTGCCTGGTTCGGCTGGGCCTAT

GACCTGAAGTCTGTGAGCGAGGATATCATCAAGCAGAGGGCAAAGAGGACAGGCGATGGCA

GCTCCGGCGTGATCTGGGGATGGGACGATAAGGATATGGACAGAGATATCAAGTCCAAGGC

CAATATCTTCTACGCCAAGAAGGAGTGA

SEQ ID NO: 29

Y. lipolytica OLE1 leader - H. zea Z11 desaturase Homo

sapiens optimized

ATGGTGAAAAACGTGGACCAAGTGGATCTCTCGCAGGTCGACACCATTGCCTCCGGCCGAG

ATGTCAACTACAAGGTCAAGTACACCTCCGGCGTTCGCAAGTATCAGATCGTGTATCCTAA

CCTGATCACCTTCGGCTACTGGCATATCGCTGGACTGTACGGACTGTATCTGTGCTTCACT

TCCGCCAAGTGGGCCACCATCCTGTTCTCTTACATCCTGTTTGTGCTGGCAGAGATCGGAA

TCACCGCAGGAGCACACAGACTGTGGGCACACAAGACATATAAGGCCAAGCTGCCACTGGA

GATCCTGCTGATGGTGTTCAACAGCATCGCCTTTCAGAATTCCGCCATCGATTGGGTGCGG

GACCACAGACTGCACCACAAGTACTCCGACACAGATGCCGACCCCCACAACGCCTCTAGGG

GCTTCTTTTATAGCCACGTGGGATGGCTGCTGGTGCGGAAGCACCCTGAGGTGAAGAAGAG

AGGCAAGGAGCTGAATATGTCTGATATCTACAACAATCCTGTGCTGCGCTTCCAGAAGAAG

TATGCCATCCCATTCATCGGCGCCGTGTGCTTTGCCCTGCCCACCATGATCCCCGTGTACT

TTTGGGGCGAGACATGGAGCAACGCCTGGCACATCACAATGCTGCGGTATATCATGAACCT

GAATGTGACATTCCTGGTGAACTCCGCCGCCCACATCTGGGGCAATAAGCCATACGACGCC

AAGATCCTGCCCGCCCAGAACGTGGCCGTGAGCGTGGCAACCGGAGGAGAGGGCTTCCACA

ATTACCACCACGTGTTTCCATGGGATTATAGGGCAGCAGAGCTGGGAAACAATTCTCTGAA

TCTGACCACAAAGTTCATCGACCTGTTTGCCGCCATCGGCTGGGCCTATGATCTGAAGACA

GTGAGCGAGGACATGATCAAGCAGAGGATCAAGCGCACCGGCGATGGCACAGACCTGTGGG

GGCACGAGCAGAATTGTGATGAAGTGTGGGATGTGAAGGATAAAAGCAGTTGA

SEQ ID NO: 30
Native A. transitella Z11 desaturase

ATGGTCCCTAACAAGGGTTCCAGTGACGTTTTGTCTGAACATTCTGAGCCCCAGTTCACTA

AACTCATAGCTCCACAAGCAGGGCCGAGGAAATACAAGATAGTGTATCGAAATTTGCTCAC

ATTCGGCTATTGGCACTTATCAGCTGTTTATGGGCTCTACTTGTGCTTTACTTGTGCGAAA

TGGGCTACCATCTTATTTGCATTTTTCTTATACGTGATCGCGGAAATCGGTATAACAGGTG

GCGCTCATAGGCTATGGGCACATCGGACTTATAAAGCCAAGTTGCCTTTAGAGATTTTGTT

ACTCATAATGAATTCTATTGCCTTCCAAGACACTGCTTTCACCTGGGCTCGAGATCACCGC

CTTCATCACAAATATTCGGATACTGACGCTGATCCCCACAATGCTAGCAGAGGGTTTTTCT

ATTCACATGTAGGCTGGCTTTTGGTGAAGAAACACCCTGAAGTCAAAGCAAGAGGAAAATA

CTTGTCGTTAGATGATCTTAAGAATAATCCATTGCTTAAATTCCAAAAGAAATACGCTATT

CTAGTTATAGGCACGTTATGCTTCCTTATGCCAACATTTGTGCCCGTATACTTCTGGGGCG

AGGGCATCAGCACGGCCTGGAACATCAATCTATTGCGATACGTCATGAATCTTAACATGAC

TTTCTTAGTTAACAGTGCAGCGCATATCTTTGGCAACAAACCATACGATAAGAGCATAGCC

TCAGTCCAAAATATTTCAGTTAGCTTAGCTACTTTTGGCGAAGGATTCCATAATTACCATC

ACACTTACCCCTGGGATTATCGTGCGGCAGAATTAGGAAATAATAGGCTAAATATGACTAC

TGCTTTCATAGATTTCTTCGCTTGGATCGGCTGGGCTTATGACTTGAAGTCTGTGCCACAA

GAGGCGATTGCAAAAAGGTGTGCGAAAACTGGCGATGGAACGGATATGTGGGGTCGAAAAA

GATAA

SEQ ID NO: 31
pPV0228_-_Z11_Helicoverpa zea_desaturase

ATGGCCCAAAGCTATCAATCAACTACGGTTTTGAGTGAGGAGAAAGAACTAACATTACAAC

ATTTGGTGCCCCAAGCATCGCCCAGGAAGTATCAAATAGTGTATCCGAACCTCATTACGTT

TGGTTACTGGCACATAGCCGGACTTTATGGCCTTTACTTGTGCTTCACTTCTGCTAAATGG

GCTACGATTTTATTCAGCTACATCCTCTTCGTGTTAGCAGAAATAGGAATCACGGCTGGCG

CTCACAGACTCTGGGCCCACAAAACTTACAAAGCGAAACTACCATTAGAAATACTCTTAAT

GGTATTCAACTCCATCGCTTTTCAAAACTCAGCCATTGACTGGGTGAGGGACCACCGACTC

CACCATAAGTATAGCGATACAGATGCTGATCCCCACAATGCCAGCCGAGGGTTCTTTTATT

CCCATGTAGGATGGCTACTTGTGAGAAAACATCCTGAAGTCAAAAAGCGAGGGAAAGAACT

CAATATGTCCGATATTTACAACAATCCTGTCTTACGGTTTCAGAAAAAATACGCCATACCC

TTCATTGGGGCTGTTTGTTTCGCCTTACCTACAATGATACCTGTTTAGTTCTGGGGAGAAA

CCTGGTCCAATGCTTGGCATATCACCATGCTTCGCTACATCATGAACCTCAATGTCACCTT

TTTGGTAAACAGCGCTGCTCATATATGGGGAAACAAGCCTTATGACGCAAAAATATTACCT

GCACAAAATGTAGCTGTGTCGGTCGCCACTGGTGGAGAAGGTTTCCATAATTACCACCATG

TCTTCCCCTGGGATTATCGAGCAGCGGAACTCGGTAACAATAGCCTCAATTTAACGACTAA

ATTCATAGATTTATTCGCAGCAATCGGATGGGCATATGATTTAAAGACGGTTTCGGAGGAT

ATGATAAAACAAAGGATTAAACGCACTGGAGATGGAACGGATCTTTGGGGACACGAACAAA

ACTGTGATGAAGTGTGGGATGTAAAAGATAAATCAAGTTAA

SEQ ID NO: 32
pPV0228_-_Helicoverpa armigera reductase codon optimized

ATGGTCGTTTTAACTTCTAAAGAGACAAAACCTTCAGTAGCTGAGTTTTATGCGGGAAAAT

CTGTTTTTATTACGGGTGGCACTGGATTCCTTGGAAAGGTATTCATAGAGAAACTTTTATA

TAGCTGTCCAGATATCGAGAATATCTACATGCTCATACGAGAGAAGAAAGGTCTTTCTGTT

AGCGAAAGAATAAAACAGTTCCTTGATGACCCGCTCTTTACCAGACTAAAAGACAAAAGAC

CAGCTGACTTAGAGAAGATTGTATTAATACCAGGAGATATTACTGCTCCTGACTTAGGCAT

TAATTCTGAAAACGAGAAGATGCTTATAGAGAAGGTATCGGTGATTATTCATTCGGCTGCT

ACGGTGAAGTTTAATGAGCCTCTCCCTACGGCTTGGAAGATCAACGTGGAAGGAACCAGAA

TGATGTTAGCTTTGAGTCGAAGAATGAAGCGGATTGAGGTTTTCATTCACATATCGACAGC

ATACACGAACACAAACAGGGAAGTGGTTGACGAGATCTTATACCCAGCTCCTGCTGATATC

GACCAAGTTCATCAGTATGTCAAAGATGGAATCTCTGAGGAAGACACTGAGAAAATATTAA

ATGGTCGTCCAAATACGTACACGTTTACGAAAGCGTTAACTGAGCATTTAGTTGCTGAGAA

CCAAGCCTACGTACCCACTATTATCGTCAGGCCGTCAGTCGTGGCAGCAATAAAAGATGAG

CCATTAAAAGGTTGGTTAGGCAACTGGTTTGGAGCGACTGGTCTCACCGTGTTCACCGCTA

AGGGTCTCAACCGAGTCATCTACGGTCATTCTAGCTACATCGTAGACTTAATTCCTGTGGA

TTATGTCGCTAATTTAGTGATTGCTGCTGGGGCTAAGAGTAGCAAGTCAACTGAGTTGAAG

GTATAGAACTGCTGCAGCAGCTCCTGCAATCCCGTCACTATTGGCACGTTAATGAGCATGT

TTGCTGACGATGCCATCAAACAGAAGTCGTATGCTATGCCGCTACCGGGGTGGTACATATT

CACGAAATATAAGTGGTTAGTTCTTCTTTTAACATTTCTCTTCCAAGTTATACCGGCGTAT

GTCACAGATCTCTCCAGGCACTTGATTGGGAAGAGTCCACGGTACATAAAACTCCAATCAC

TAGTAAATCAAACGCGCTCTTCAATCGACTTCTTCACGAATCACTCCTGGGTGATGAAGGC

AGACAGAGTGAGAGAGTTATATGCGTCTCTTTCCCCCGCAGACAAGTACTTATTTCCCTGT

GATCCTACGGACATTAACTGGACACATTACATACAAGACTACTGTTGGGGAGTCCGACATT

TTTTGGAGAAAAAAAGCTACGAATAA

SEQ ID NO: 33
pPV0228_-_ICL_promoter

TATTAGGCGAAGAGGCATCTAGTAGTAGTGGCAGTGGTGAGAACGTGGGCGCTGCTATAGT

GAACAATCTCCAGTCGATGGTTAAGAAGAAGAGTGACAAACCAGCAGTGAATGACTTGTCT

GGGTCCGTGAGGAAAAGAAAGAAGCCCGACACAAAGGACAGTAACGTCAAGAAACCCAAGA

AATAGGGGGGACCTGTTTAGATGTATAGGAATAAAAACTCCGAGATGATCTCAATGTGTAA

TGGAGTTGTAATATTGCAAAGGGGGAAAATCAAGACTCAAACGTGTGTATGAGTGAGCGTA

CGTATATCTCCGAGAGTAGTATGACATAATGATGACTGTGAATCATCGTAATCTCACACAA

AAACCCCATTGTCGGCCATATACCACACCAAGCAACACCACATATCCCCCGGAAAAAAAAA

CGTGAAAAAAAGAAACAATCAAAACTACAACCTACTCCTTGATCACACAGTCATTGATCAA

GTTACAGTTCCTGCTAGGGAATGACCAAGGTACAAATCAGCACCTTAATGGTTAGCACGCT

CTCTTACTCTCTCTCACAGTCTTCCGGCCCCTATTCAAAATTCTGCACTTCCATTTGACCC

CAGGGTTGGGAAACAGGGCCACAAAAGAAAAACCCGACGTGAATGAAAAAACTAAGAAAAG

AAAAAAAATTATCACACCAGAAATTTACCTAATTGGGTAATTCCCATCGGTGTTTTTCCTG

GATTGTCGCACGCACGCATGCTGAAAAAAGTGTTCGAGTTTTGCTTTTGCCTCGGAGTTTC

ACGCAAGTTTTTCGATCTCGGAACCGGAGGGCGGTCGCCTTGTTGTTTGTGATGTCGTGCT

TTGGGTGTTCTAATGTGCTGTTATTGTGCTCTTTTTTTTTCTTCTTTTTTTGGTGATCATA

TGATATTGCTCGGTAGATTACTTTCGTGTGTAGGTATTCTTTTAGACGTTTGGTTATTGGG

TAGATATGAGAGAGAGAGAGTGGGTGGGGGAGGAGTTGGTTGTAGGAGGGACCCCTGGGAG

GAAGTGTAGTTGAGTTTTCCCTGACGAATGAAAATACGTTTTTGAGAAGATAATACAGGAA

AGGTGTGTCGGTGAATTTCCATCTATCCGAGGATATGAGTGGAGGAGAGTCGTGTGCGTGT

GGTTAATTTAGGATCAGTGGAACACACAAAGTAACTAAGACAGAGAGACAGAGAGAAAAAT

CTGGGGAAGAGACAAAGAGTCAGAGTGTGTGAGTTATTCTGTATTGTGAAATTTTTTTGCC

CAACTACATAATATTGCTGAAACTAATTTTACTTAAAAAGAAAAGCCAACAACGTCCCCAG

TAAAACTTTTCTATAAATATCAGCAGTTTTCCCTTTCCTCCATTCCTCTTCTTGTCTTTTT

TCTTACTTTCCCTTTTTTATACCTTTTCATTATCATCCTTTATAATTGTCTAACCAACAAC

TATATATCTATCAA

SEQ ID NO: 34
pPV0228_-_TEF_Candida tropicalis_promoter_region

AGGAAGACAACCAAAAGAAAGATCAAATTGACTAAATGTTGAACAGACCAAAAAAAAAGAA

CAACAAATAGATAAATTACAACATATTAATCTTTTGATATGTTGTTGAATATTCTAGTAAA

TCTAATGATCTCAATAGTGGTTATCATTCACTCTCTTCGTCCTCCTCTCTCCCCTCCTCCT

CTTGCAGTATATTAAAAGCAATAAAAAAAAAAAAAAAAAGAAAATCTGCCAACACACACAA

AAAAAACTTACATAGTCGTGTACCAGTGTCAATATTTCACCAGCGCAGAGAAAAGAAGATG

AACAGAAAAATTTTCTCTTTGGTTTTGTCTTTGGTTTTGTATTAATCTCATTGAAAAATTT

TTTCTCTCTCTCTCTCTCTCTCTCTCACTCACACACTCACTCGCATTTCGTTTGGGTTACA

GCAGAAGTCAGACAGAAAAAAAAAATCGTATATAACTCTCATCAAATGCCCTAGAGAAAAA

TTTTTCTTCTATCCTTTTTTTTTTCTTCTTCTTCTTCTTTTCCTTTTTTCTTTTAGAAGAT

CTTTTTGAATTCATCAAAGATATATATTTAATCAATC

SEQ ID NO: 35
pPV0228_-_ICL_terminator

AAGAAAAAACAAAAGGTAAAGAACTTCATTTGAGATGAACTTTTGTATATGACTTTTAGTT

TCTAGTTTTTTTTTTATTTATTGCTTAATTTTCTTTATTTCAATCCCCCATAGTTTGTGTA

GAATATATTTATTCATTCTGGTAACTCAAACACGTAGCAAGCTCGTTGCATCTCGCCTCGT

CACGGGTACAGCTCTGGAACCAAAGACAAAAAAAAAAGTTGATCCGAACCCTCTCGCTATT

CCTTGCTATGCTATCCACGAGATGGGGTTTATCAGCCCAGGCAAGTCACTAAA

SEQ ID NO: 36
pPV0228_-_TEF_terminator

GCTGATTAATGAATAATTAATAAGTATTGTTTTTTTTGTTTTTAATATATATATATCTTGA

AATTAGTATAAAAAAAATCTTTTTTTTTTCTTTTTTATTTATTTTATCAATAGTTTATATA

TATATATATATAAACTTGTAAGAGATTAGGTATATCTAACAGTGATACTACTAATAGTGCT

TAATATCTTTGTTAAACAAGAAAATAAAATAAAC

SEQ ID NO: 37
SapI-tLIP2-pEXP1-HA_FAR-SapI (insert into pPV199 creating

pPV247)

GCCTGAAGAGCGCTATTTATCACTCTTTACAACTTCTACCTCAACTATCTACTTTAATAAA

TGAATATCGTTTATTCTCTATGATTACTGTATATGCGTTCCTCCATGGGAGTTTGGCGCCC

GTTTTTTCGAGCCCCACACGTTTCGGTGAGTATGAGCGGCGGCAGATTCGAGCGTTTCCGG

TTTCCGCGGCTGGACGAGAGCCCATGATGGGGGCTCCCACCACCAGCAATCAGGGCCCTGA

TTACACACCCACCTGTAATGTCATGCTGTTCATCGTGGTTAATGCTGCTGTGTGCTGTGTG

TGTGTGTTGTTTGGCGCTCATTGTTGCGTTATGCAGCGTAGACCACAATATTGGAAGCTTA

TTAGCCTTTCTATTTTTTCGTTTGCAAGGCTTAACAACATTGCTGTGGAGAGGGATGGGGA

TATGGAGGCCGCTGGAGGGAGTCGGAGAGGCGTTTTGGAGCGGCTTGGCCTGGCGCCCAGC

TCGCGAAACGCACCTAGGACCCTTTGGCACGCCGAAATGTGCCACTTTTCAGTCTAGTAAC

GCCTTACCTACGTCATTCCATGCATGCATGTTTGCGCCTTTTTTCCCTTGCCCTTGATCGC

CACACAGTACAGTGCACTGTACAGTGGAGGTTTTGGGGGGGTCTTAGATGGGAGCTAAAAG

CGGCCTAGCGGTACACTAGTGGGATTGTATGGAGTGGCATGGAGCCTAGGTGGAGCCTGAC

AGGACGCACGACCGGCTAGCCCGTGACAGACGATGGGTGGCTCCTGTTGTCCACCGCGTAC

AAATGTTTGGGCCAAAGTCTTGTCAGCCTTGCTTGCGAACCTAATTCCCAATTTTGTCACT

TCGCACCCCCATTGATCGAGCCCTAACCCCTGCCCATCAGGCAATCCAATTAAGCTCGCAT

TGTCTGCCTTGTTTAGTTTGGCTCCTGCCCGTTTCGGCGTCCACTTGCACAAACACAAACA

AGCATTATATATAAGGCTCGTCTCTCCCTCCCAACCACACTCACTTTTTTGCCCGTCTTCC

CTTGCTAACACAAAAGTCAAGAACACAAACAACCACCCCAACCCCCTTACACACAAGACAT

ATCTACAGCAATGGTGGTGCTGACCAGCAAGGAGACAAAGCCTTCCGTGGCCGAGTTCTAC

GCCGGCAAGTCCGTGTTTATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGA

AGCTGCTGTACTCTTGCCCAGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGG

CCTGAGCGTGTCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACACGGCTGAAG

GACAAGAGACCTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAG

ATCTGGGCATCAACTCCGAGAATGAGAAGATGCTGATCCAGAAGGTGTCCGTGATCATCCA

CTCTGCCGCCACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAG

GGCACCAGGATGATGCTGGCCCTGAGCCGGAGAATGAAGCGCATCGAGGTGTTTATCCACA

TCTCCACAGCCTACACCAACACAAATCGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCC

CGCCGACATCGATCAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAG

AAGATCCTGAACGGCCGGCCAAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGG

TGGCCGAGAACCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCAT

CAAGGATGAGCCCCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTG

TTTACAGCCAAGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTGA

TCCCCGTGGATTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGTCTAGCAAGTCTAC

CGAGCTGAAGGTGTATAACTGCTGTTCCTCTAGCTGTAATCCTGTGACCATCGGCACACTG

ATGTCCATGTTCGCCGACGATGCCATCAAGCAGAAGTCTTACGCCATGCCTCTGCCAGGCT

GGTACATCTTTACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCAT

CCCAGCCTACGTGACCGATCTGTCTAGGCACCTGATCGGCAAGAGCCCCCGCTATATCAAG

CTGCAGTCTCTGGTGAACCAGACGAGGTCCTCTATCGACTTCTTTACAAATGACAGCTGGG

TCATGAAGGCCGATAGGGTGCGCGAGCTGTACGCCTCTCTGAGCCCTGCCGACAAGTATCT

GTTCCCCTGCGACCCTACCGATATCAATTGGACACACTACATCCAGGATTATTGTTGGGGC

GTGCGCCACTTCCTGGAGAAGAAGTCCTATGAGTGAGCCTGAAGAGC

SEQ ID NO: 38
NcoI-pTAL-AleI (insert into pPV247 creating pPV248)

CCATGGGTAAGCAGGTGGCTCCGTTTGTGTCTTTGTGTTTTTCCCCTCCTTTTTGGACCAT

TTGTCAGCATGTTGCGTAGGTCTGGGTGTTTGACTGTTCAGGTGGTGGATGACGGATGCAT

CATCTGACGGCAGAGTGGGTACCTGGCAGTGGCAGGCTCGCAGACGAGGTAGAGAGATTCT

GAAAGGAGCCATTGACAGATGGAGAATTGGATACTCCTGGTATGTCCTCCGTTTCCACTTT

TGACGTTGGTGACGTGCTCTGGAACGACTTTTTTCTTTTTCTTTAAAACAAAAAAAAGAAA

GAAAAAAAAAACATTTACTACTACCAGTAGTACACCTCAACATTGGGTCCAGAACGTCCCA

ACTGCATGAGTCACTGGAGTCATGCCGAGGTCGCTAAGGTGCTGTAAAATACAACGTCAAT

TGAGAGAGACACAGGCGCAGCGCGCCGAGGGAGAAACGAGGCATTTATCTTCTGACCCTCC

TTTTTACTCGTAATCTGTATCCCGGAACCGCGTCGCATCCATGTTAATTAAATCAACACTT

ACACTTGCTTGCTTCGTATGATGAAGATTTCTGACTGGCAACCCAGTCAGCAGCAGATTGG

GGCAGATGTAGTAATGAAAAACACTGCAAGGTGTGACGTTTGAGACACTCCAATTGGTTAG

AAAGCGACAAAGAAGACGTCGGAAAAATACCGGAAAAATCGAGTCTTTTTCTTTCTGCGTA

TTGGGCCCTTCTGCCTCCTTTGCCGCCCTTTCCACGCTCTTTCCACACCCTCACACTCCCT

GAGCACTATGATCTCATTGCGCAATAAGATATACATGCACGTGCATTTGGTGAGCACGCAG

AACCTTGTTGGGGGAAGATGCCCTAACCCTAAGGGCGTTCCATACGGTTCGACAGAGTAAC

CTTGCTGTCGATTATAACGCATATATAGCCCCCCCCTTCGGACCCTCCTTCTGATTTCTGT

TTCTGTATCAACATTACACACAAACACACAATGGTG

SEQ ID NO: 39
pDST003_Sesamia inferens desaturase

MLSQEEPTDTSLVPRAAPRKYQIVYPNLITFGYWHLAGLYGLYLCFTSAKWTTILFSFILC

VIAEIGVTAGAHRLWAHKTYKANLPLQILLMVMNSIAFQNSAIDWVPDHRLHHKYSDTDAD

PHNASRGFFYSHVGWLLVKKHPEVKKRGKELDMSDIYSNPVLRFQKQYAIPFIGAVCFILP

TVIPVYCWGETWTNAWHITMLRYITNLNVTFLVNSAAHIWGYKPYDENILPAQNIAVSIAT

CGEGFHNYHHVFPWDYRAAELGNNNLNLTTKFIDFFAWLGWAYDLKTVSSDMIKLRAKRTG

DGTNLWGEHNDELKEGKED

SEQ ID NO: 40

H. armigera FAR from SEQ ID NO: 37

ATGGTGGTGCTGACCAGCAAGGAGACAAAGCCTTCCGTGGCCGAGTTCTAGGCCGGCAAGT

CCGTGTTTATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTA

CTCTTGCCCAGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGGCCTGAGCGTC

TCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACACGGCTGAAGGACAAGAGAC

CTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAGATCTGGGCAT

CAACTCCGAGAATGAGAAGATGCTGATCGAGAAGGTGTCCGTGATCATCCACTCTGCCGCC

ACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGA

TGATGCTGGCCCTGAGCCGGAGAATGAAGCGCATCGAGGTCTTTATCCACATCTCCACAGC

CTACACCAACACAAATCGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATC

GATCAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAGAAGATCCTGA

ACGGCCGGCCAAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGGTGGCCGAGAA

CCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAG

CCCCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCCA

AGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTGATCCCCGTGGA

TTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGTCTAGCAAGTCTACCGAGCTGAAG

GTGTATAACTGCTGTTCCTCTAGCTGTAATCCTGTGACCATCGGCACACTGATGTCCATGT

TCGCCGACGATGCCATGAAGCAGAAGTCTTACGCCATGCCTCTGCCAGGCTGGTACATCTT

TACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCATCCCAGCCTAC

GTGACCGATCTGTCTAGGCACCTGATCGGCAAGAGCCCCCGCTATATCAAGCTGCAGTCTC

TGGTGAACCAGACCAGGTCCTCTATCGACTTCTTTACAAATCACAGCTGGGTCATGAAGGC

CGATAGGGTGCGCGAGCTGTACGCCTCTCTGAGCCCTGCCGACAAGTATCTGTTCCCCTGC

GACCCTACCGATATCAATTGGACACACTACATCCAGGATTATTGTTGGGGCGTGCGCCACT

TCCTGGAGAAGAAGTCCTATGAGTGA

SEQ ID NO: 41

H. armigera alcohol forming reductase (HaFAR)

MVVLTSKETKPSVAEFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIENIYMLIREKKGLS

VSERIKQFLDDPLFTRLKDKRPADLEKIVLIPGDITAPDLGINSENEKMLIEKVSVIIHS

AATVKFNEFLPTAWKINVEGTRMMLALSRRMKRIEVFIHISTAYTNTNREVVDEILYPAP

ADIDQVHQYVKDGISEEDTEKILNGRPNTYTFTKALTEHLVAENQAYVPTIIVRPSVVAA

IKDEPLKGWLGNWFGATGLTVFTAKGLNRVIYGHSSYIVDLIPVDYVANLVIAAGAKSSK

STELKVYNCCSSSCNPVTIGTLMSMFADDAIKQKSYAMPLPGWYIFTKYKWLVLLLTFLF

QVIPAYVTDLSRHLIGKSPRYIKLQSLVNQTRSSIDFFTNHSWVMKADRVRELYASLSPA

DKYLFPCDPTDINWTHYIQDYCWGVRHFLEKKSYE

SEQ ID NO: 42
HaFAR S60A

MVVLTSKETKPSVAEFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIENIYMLIREKKGLAV

SERIKQFLDDFLFTRLKDKRPADLEKIVLIPGDITAPDLGINSENEKMLIEKVSVIIHSAA

TVKFNEPLPTAWKINVEGTRMMLALSRRMKRIEVFIHISTAYTNTNREVVDEILYPAPADI

DQVHQYVKDGISEEDTEKILNGRPNTYTFTKALTEHLVAENQAYVPTIIVRPSVVAAIKDE

PLKGWLGNWFGATGLTVFTAKGLNRVIYGHSSYIVDLIPVDYVANLVIAAGAKSSKSTELK

VYNCCSSSCNPVTIGTLMSMFADDAIKQKSYAMPLPGWYIFTKYKWLVLLLTFLFQVIPAY

VTDLSRHLIGKSPRYIKLQSLVNQTRSSIDFFTNHSWVMKADRVRELYASLSPADKYLFPC

DPTDINWTHYIQDYCWGVRHFLEKKSYE

SEQ ID NO: 43
HaFAR S195A

MVVLTSKETKPSVAEFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIENIYMLIREKKGLSV

SERIKQFLDDPLFTRLKDKRPADLEKIVLIPGDITAPDLGINSENEKMLIEKVSVIIHSAA

TVKFNEPLPTAWKINVEGTRMMLALSRRMKRIEVFIHISTAYTNTNREVVDEILYPAPADI

DQVHQYVKDGIAEEDTEKILNGRPNTYTFTKALTEHLVAENQAYVPTIIVRPSVVAAIKDE

PLKGWLGNWFGATGLTVFTAKGLNRVIYGHSSYIVDLIPVDYVANLVIAAGAKSSKSTELK

VYNCCSSSCNPVTIGTLMSMFADDAIKQKSYAMPLPGWYIFTKYKWLVLLLTFLFQVIPAY

VTDLSRHLIGKSPRYIKLQSLVNQTRSSIDFFTNHSWVMKADRVRELYASLSPADKYLFPC

DPTDINWTHYIQDYCWGVRHFLEKKSYE

SEQ ID NO: 44
HaFAR S298A

MVVLTSKETKPSVAEFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIENIYMLIREKKGLSV

SERIKQFLDDPLFTRLKDKRPADLEKIVLIPGDITAPDLGINSENEKMLIEKVSVIIHSAA

TVKFNEPLPTAWKINVEGTRMMLALSRRMKRIEVFIHISTAYTNTNREVVDEILYPAPADI

DQVHQYVKDGISEEDTEKILNGRPNTYTFTKALTEHLVAENQAYVPTIIVRPSVVAAIKDE

PLKGWLGNWFGATGLTVFTAKGLNRVTYGHSSYIVDLIPVDYVANLVIAAGAKASKSTELK

VYNCCSSSCNPVTIGTLMSMFADDAIKQKSYAMPLPGWYIFTKYKWLVLLLTFLFQVIPAY

VTDLSPHLIGKSPRYIKLQSLVNQTRSSIDFFTNHSWVMKADRVRELYASLSPADKYLFPC

DPTDINWTHYIQDYCWGVRHFLEKKSYE

SEQ ID NO: 45
HaFAR S378A

MVVLTSKETKPSVAEFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIENIYMLIREKKGLSV

SERIKQFLDDPLFTRLKDKRPADLEKIVLIPGDITAPDLGINSENEKMLIEKVSVIIHSAA

TVKFNEPLPTAWKINVEGTRMMLALSRRMKRIEVFIHISTAYTNTNREVVDEILYPAPADI

DQVHQYVKDGISEEDTEKILNGRPNTYTFTKALTEHLVAENQAYVPTIIVRPSVVAAIKDE

PLKGWLGNWFGATGLTVFTAKGLNRVIYGHSSYIVDLIPVDYVANLVIAAGAKSSKSTELK

VYNCCSSSCNPVTIGTLMSMFADDAIKQKSYAMPLPGWYIFTKYKWLVLLLTFLFQVIPAY

VTDLSRHLIGKAPRYIKLQSLVNQTRSSIDFFTNHSWVMKADRVRELYASLSPADKYLFPC

DPTDINWTHYIQDYCWGVRHFLEKKSYE

SEQ ID NO: 46
HaFAR S394A

MVVLTSKETKPSVAEFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIENIYMLIREKKGLSV

SERIKQFLDDPLFTRLKDKRPADLEKIVLIPGDITAPDLGINSENEKMLIEKVSVIIHSAA

TVKFNEPLPTAWKINVEGTRMMLALSRRMKRIEVFIHISTAYTNTNREVVDEILYPAPADI

DQVHQYVKDGISEEDTEKILNGRPNTYTFTKALTEHLVAENQAYVPTIIVRPSVVAAIKDE

PLKGWLGNWFGATGLTVFTAKGLNRVIYGHSSYIVDLIPVDYVANLVIAAGAKSSKSTELK

VYNCCSSSCNPVTIGTLMSMFADDAIKQKSYAMPLPGWYIFTKYKWLVLLLTFLFQVIPAY

VTDLSRHLIGKSPRYIKLQSLVNQTRSAIDFFTNHSWVMKADRVRELYASLSPADKYLFPC

DPTDINWTHYIQDYCWGVRHFLEKKSYE

SEQ ID NO: 47
HaFAR S418A

MVVLTSKETKPSVAEFYAGKSVFITGGTGFLCKVFIEKLLYSCPDIENIYMLIREKKGLSV

SERIKQFLDDPLFTRLKDKRPADLEKIVLIPGDITAPDLGINSENEKMLIEKVSVIIHSAA

TVKFNEPLPTAWKINVEGTRMMLALSRRMKRIEVFIHISTAYTNTNREVVDEILYPAPADI

DQVHQYVKDGISEEDTEKILNGRPNTYTFTKALTEHLVAENQAYVPTIIVRPSVVAAIKDE

PLKGWLGNWFGATGLTVFTAKGLNRVIYGHSSYIVDLIPVDYVANLVIAAGAKSSKSTELK

VYNCCSSSCNPVTIGTLMSMFADDAIKQKSYAMPLPGWYIFTKYKWLVLLLTFLFQVIPAY

VTDLSRHLIGKSPRYIKLQSLVNQTRSSIDFFTNHSWVMKADRVRELYASLAPADKYLFPC

DPTDINWTHYIQDYCWGVRHFLEKKSYE

SEQ ID NO: 48
HaFAR S453A

MVVLTSKETKPSVAEFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIENIYMLIREKKGLSV

SERIKQFLDDPLFTRLKDKRPADLEKIVLIPGDITAPDLGINSENEKMLIEKVSVIIHSAA

TVKFNEPLPTAWKINVEGTRMMLALSRRMKRIEVFIHISTAYTNTNREVVDEILYPAPADI

DQVHQYVKDGISEEDTEKILNGRPNTYTFTKALTEHLVAENQAYVPTIIVRPSVVAAIKDE

PLKGWLGNWFGATGLTVFTAKGLNRVIYGHSSYIVDLIPVDYVANLVIAAGAKSSKSTELK

VYNCCSSSCNPVTIGTLMSMFADDAIKQKSYAMPLPGWYIFTKYKWLVLLLTFLFQVIPAY

VTDLSRHLIGKSPRYIKLQSLVNQTRSSIDFFTNHSWVMKADRVRELYASLSPADKYLFPC

DPTDINWTHYIQDYCWGVRHFLEKKAYE

SEQ ID NO: 49

Trichoplusia ni desaturase

MAVMAQTVQETATVLEEEARTVTLVAPKTTPRKYKYIYTNFLTFSYAHLAALYGLYLCFTS

AKWETLLFSFVLFHMSNIGITAGAHRLWTHKTFKAKLPLEIVLMIFNSLAFQNTAITWARE

HRLHHKYSDTDADPHNASRGFFYSHVGWLLVKKHPDVLKYGKTIDMSDVYNNPVLKFQKKY

AVPLIGTVCFALPTLIPVYCWGESWNNAWHIALFRYIFNLNVTFLVNSAAHIWGNKPYDKS

ILPAQNLLVSFLASGEGFHNYHHVFPWDYRTAELGNNFLNLTTLFIDFCAWFGWAYDLKSV

SEDIIKQRAKRTGDGSSGVIWGWDDKDMDRDIKSKANIFYAKKE

SEQ ID NO: 50

T. pseudonana desaturase encoded by SEQ ID NO: 11

TSMDFLSGDPFRTLVLAALVVIGFAAAWQCFYPPSIVGKPRTLSNGKLNTRIHGKLYDLSS

FQHPGGPVALSLVQGRDGTALFESHHPFIPRKNLLQILSKYEVPSTEDSVSFIATLDELNG

ESPYDWKDIENDDFVSDLRALVIEHFSPLAKERGVSLVESSKATPQRWMVVLLLLASFFLS

IPLYLSGSWTFVVVTPILAWLAVVNYWHDATHFALSSNWILNAALPYLLPLLSSPSMWYHH

HVIGHHAYTNISKRDPDLAHAPQLMREHKSIKWRPSHLNQTQLPRILFIWSIAVGIGLNLL

NDVRALTKLSYNNVVRVEKMSSSRTLLHFLGRMLHIFVTTLWPFLAFPVWKAIVWATVPNA

ILSLCFMLNTQINHLINTCAHASDNNFYKHQVVTAQNFGRSSAFCFIFSGGLNYQIEHHLL

PTVNHCHLPALAPGVERLCKKHGVTYNSVEGYREAIIAHFAHTKDMSTKPTD

SEQ ID NO: 51

T. pseudonana Z11 desaturase encoded by SEQ ID NO: 23

MDFLSGDPFRTLVLAALVVIGFAAAWQCFYPPSIVGKPRTLSNGKLNTRIHGKLYDLSSFQ

HPGGPVALSLVQGRDGTALFESHHPFIPRKNLLQILSKYEVPSTEDSVSFIATLDELNGES

PYDWKDIENDDFVSDLRALVIEHFSPLAKERGVSLVESSKATPQRWMVVLLLLASFFLSIP

LYLSGSWTFVVVTPILAWLAVVNYWHDATHFALSSNWILNAALPYLLPLLSSPSMWYHHHV

IGHHAYTNISKRDPDLAHAPQLMREHKSIKWRPSHLNQTQLPRILFIWSIAVGIGLNLLND

VRALTKLSYNNVVRVEKMSSSRTLLHFLGRMLHIFVTTLWPFLAFPVWKAIVWATVPNAIL

SLCFMLNTQINHLINTCAHASDNNFYKHQVVTAQNFGRSSAFCFIFSGGLNYQIEHHLLPT

VNHCHLPALAPGVERLCKKHGVTYNSVEGYREAIIAHFAHTKDMSTKPTD

SEQ ID NO: 52

Amyelois transitella desaturase

MVPNKGSSDVLSEHSEPQFTKLIAPQAGPRKYKIVYRNLLTFGYWHLSAVYGLYLCFTCAK

WATILFAFFLYVIAEIGITGGAHRLWAHRTYKAKLPLEILLLIMNSIAFQDTAFTWARDHR

LHKKYSDTDADPHNATRGFFYSHVGWLLVKKHPEVKARGKYLSLDDLKNNPLLKFQKKYAI

LVIGTLCFLMPTFVPVYFWGEGISTAWNINLLRYVMNLNMTFLVNSAAHIFGNKPYDKSIA

SVQNISVSLATFGEGFHNYHHTYPWDYRAAELGNNRLNMTTAFIDFFAWIGWAYDLKSVPQ

EAIAKRCAKTGDGTDMWGRKR

SEQ ID NO: 53

Agrotis segetum desaturase

MAQGVQTTTILREEEPSLTFVVPQEPRKYQIVYPNLITFGYWHIAGLYGLYLCFTSAKWQT

ILFSFMLVVLAELGITAGAHRLWAHKTYKAKLPLQIILMILNSIAFQNSAIDWVRDHRLHH

KYSDTDADPHNATRGFFYSHVGWLLVRKHPEVKRRGKELDMSDIYNNPVLRFQKKYAIPFI

GAMCFGLPTFIPVYFWGETWSNAWHITMLRYILNLNITFLVNSAAHIWGYKPYDIKILPAQ

NIAVSIVTGGEVSITTTTFFPWDYRAAELGNNYLNLTTKFIDFFAWIGWAYDLKTVSSDVI

KSKAERTGDGTNLWGLEDKGEEDFLKIWKDN

SEQ ID NO: 54

Helicoverpa zea desaturase

MAQSYQSTTVLSEEKELTLQHLVPQASPRKYQIVYPNLITFGYWHIAGLYGLYLCFTSAKW

ATILFSYILFVLAEIGITAGAHRLWAHKTYKAKLPLEILLMVFNSIAFQNSAIDWVRDHRL

HHKYSDTDADPHNASRGFFYSHVGWLLVRKHPEVKKRGKELNMSDIYNNPVLRFQKKYAIP

FIGAVCFALPTMIPVYFWGETWSNAWHITMLRYIMNLNVTFLVNSAAHIWGNKPYDAKILP

AQNVAVSVATGGEGFHNYHHVFPWDYRAAELGNNSLNLTTKFIDLFAAIGWAYDLKTVSED

MIKQRIKRTGDGTDLWGHEQNCDEVWDVKDKSS

SEQ ID NO: 55

Agrotis segetum FAR encoded by SEQ ID NO: 1

MPVLTSREDEKLSVPEFYAGKSIFVTGGTGFLGKVFIEKLLYCCPDIDKIYMLIREKKNLS

IDERMSKFLDDPLFSRLKEERPGDLEKIVLIPGDITAPNLGLSAENERILLEKVSVIINSA

ATVKFNEPLPIAWKINVEGTRMLLALSRRMKRIEVFIHISTAYSNASSDRIVVDEILYPAP

ADMDQVYQLVKDGVTEEETERLLNGLPNTYTFTKALTEHLVAEHQTYVPTIIIRPSVVASI

KDEPIRGWLCNWFGATGISVFTAKGLNRVLLGKASNIVDVIPVDYVANLVIVAGAKSGGQK

SDELKIYNCCSSDCNPVTLKKIIKEFTEDTIKNKSHIMPLPGWFVFTKYKWLLTLLTIIFQ

MLPMYLADVYRVLTGKIPRYMKLHHLVIQTRLGIDFFTSHSWVMKTDRVRELFGSLSLAEK

HMFPCDPSSIDWTDYLQSYCYGVRRFLEKKK

SEQ ID NO: 56

Spodoptera littoralis FAR encoded by SEQ ID NO: 2

MVVLTSKEKSNMSVADFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIDKIYMLIREKKGQS

IRERLTKIVDDPLFNRLKDKRPDDLGKIVLIPGDITVPGLGISEENETILTEKVSVVIHSA

ATVKFNEPLATAWNVNVEGTRMIMALSRRMKRIEVFIHISTAYTNTNRAVIDEVLYPPPAD

INDVHQHVKNGVTEEETEKILNGRPNTYTFTKALTEHLVAENQSYMPTIIVRPSIVGAIKD

DPIRGWLANWYGATGLSVFTAKGLNRVIYGHSNHVVDLIPVDYVANLVIVAGAKTYHSNEV

TIYNSCSSSCNPITMKRLVGLFIDYTVKHKSYVMPLPGWYVYSNYKWLVFLVTVIFQVIPA

YLGDIGRRLLGKNPRYYKLQNLVAQTQEAVHFFTSHTWEIKSKRTSELFSSLSLTDQRMFP

CDANRIDWTDYITDYCSGYRQFLEKIK

SEQ ID NO: 57

Helicoverpa armigera FAR encoded by SEQ ID NO: 3 or 32

MVVLTSKETKPSVAEFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIENIYMLIREKKGLSV

SERIKQFLDDPLFTRLKDKRPADLEKIVLIPGDITAPDLGINSENEKMLIEKVSVIIHSAA

TVKFNEPLPTAWKINVEGTRMMLALSRRMKRIEVFIHISTAYTNTNREVVDEILYPAPADI

DQVHQYVKDGISEEDTEKILNGRPNTYTFTKALTEHLVAENQAYVPTIIVRPSVVAAIKDE

PLKGWLGNWFGATGLTVFTAKGLNRVIYGHSSYIVDLIPVDYVANLVIAAGAKSSKSTELK

VYNCCSSSCNPVTIGTLMSMFADDAIKQKSYAMPLPGWYIFTKYKWLVLLLTFLFQVIPAY

VTDLSRHLIGKSPRYIKLQSLVNQTRSSIDFFTNHSWVMKADRVRELYASLSPADKYLFPC

DPTDINWTHYIQDYCWGVRHFLEKKSYE

SEQ ID NO: 58

Ostrinia furnacalis Z9 desaturase encoded by SEQ ID NO: 20

MAPNIKDGADLNGVLFEDDASTPDYALATAPVQKADNYPRKLVWRNIILFAYLHLAAVYGA

YLFLFSAKWQTDIFAYILYVISGLGITAGAHRLWAHKSYKAKWPLRLILIIFNTVSFQDSA

LDWSRDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPELKRKGKGLDLSDLYADPIL

RFQKKYYLLLMPLGCFIMPTVVPVYFWGETWTNAFFVAALFRYTFILNVTWLVNSAAHKWG

HKPYDSSIKPSENLSVSLFALGEGFHNYHHTFPWDYKTAELGNNRLNFTTNFINFFAKIGW

AYDLKTVSDEIIQNRVKRTGDGSHHLWGWGDKDQPKEEVNAAIRINPKDE

SEQ ID NO: 59

Lampronia capitella Z9 desaturase encoded by SEQ ID NO: 21

MPPNVTEANGVLFENDVQTPDMGLEVAPVQKADERKIQLVWRNIIAFACLHLAAVYGAYLF

FTSAIWQTDIFAYILYVMSGLGITAGAHRLWAHKSYKAKWPLRLILVAFNTLAFQDSAIDW

ARDHRMHHKYSETDADPHNATRGFFFSHIGWLLCRKHPELKRKGQGLDLSDLYADPIIRFQ

KKYYLLLMPLACFVLPTIIPVYLWGESWKNAFFVAAMFRYTFILNVTWLVNSAAHKWGGKP

YDKNIQPAQNISVAIFALGEGFHNYHHTFPWDYKTAELGNNRLNFTTSFINFFASFGWAYD

LKTVSDEIIQQRVKRTGDGSHHLRGWGDQDIPAEEAQAALRINRKDD

SEQ ID NO: 60

Helicoverpa zea Z9 desaturase encoded by SEQ ID NO: 22

MAPNISEDVNGVLFESDAATPDLALSTPPVQKADNRPKQLVWRNILLFAYLHLAALYGGYL

FLFSAKWQTDIFAYILYVISGLGITAGAHRLWAHKSYKAKWPLRVILVIFNTVAFQDAAMD

WARDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPDLKEKGKGLDMSDLLADPILRF

QKKYYLILMPLACFVMPTVIPVYFWGETWTNAFFVAAMFRYAFILNVTWLVNSAAHKWGDK

PYDKSIKPSENLSVAMFALGEGFHNYHHTFPWDYKTAELGNNKLNFTTTFINFFAKIGWAY

DLKTVSDDIVKNRVKRTGDGSHHLWGWGDENQSKEEIDAAIRINPKDD

SEQ ID NO: 61

Trichoplusia ni desaturase with Yarrowia lipolytica OLE1

leader sequence encoded by SEQ ID NO: 28

MVKNVDQVDLSQVDTIASGRDVNYKVKYTSGVKTTPRKYKYIYTNFLTFSYAHLAALYGLY

LCFTSAKWETLLFSFVLFHMSNIGITAGAHRLWTHKTFKAKLPLEIVLMIFNSLAFQNTAI

TWAREHRLHHKYSDTDADPHNASRGFFYSHVGWLLVKKHPDVLKYGKTIDMSDVYNNPVLK

FQKKYAVPLIGTVCFALPTLIPVYCWGESWNNAWHIALFRYIFNLNVTFLVNSAAHIWGNK

PYDKSILPAQNLLVSFLASGEGFHNYHHVFPWDYRTAELGNNFLNLTTLFIDFCAWFGWAY

DLKSVSEDIIKQRAKRTGDGSSGVIWGWDDKDMDRDIKSKANIFYAKKE

SEQ ID NO: 62

Helicoverpa zea desaturase with Yarrowia lipolytica OLE1

leader sequence encoded by SEQ ID NO: 29

MVKNVDQVDLSQVDTIASGRDVNYKVKYTSGVRKYQIVYPNLITFGYWHIAGLYGLYLCFT

SAKWATILFSYILFVLAEIGITAGAHRLWAHKTYKAKLPLEILLMVFNSIAFQNSAIDWVR

DHRLHHKYSDTDADPHNASRGFFYSHVGWLLVRKHPEVKKRGKELNMSDIYNNPVLRFQKK

YAIPFIGAVCFALPTMIPVYFWGETWSNAWHITMLRYIMNLNVTFLVNSAAHIWGNKPYDA

KILPAQNVAVSVATGGEGFHNYHHVFPWDYRAAELGNNSLNLTTKFIDLFAAIGWAYDLKT

VSEDMIKQRIKRTGDGTDLWGHEQNCDEVWDVKDKSS

SEQ ID NO: 63

Agrotis segetum desaturase with Candida albicans OLE1

leader sequence encoded by SEQ ID NO: 15

MTTVEQLETVDITKLNAIAAGTNKKVPMAQGVQTTTILREEEPSLTFVVPQEPRKYQIVYP

NLITFGYWHIAGLYGLYLCFTSAKWQTILFSFMLVVLAELGITAGAHRLWAHKTYKAKLPL

QIILMILNSIAFQNSAIDWVRDHRLHHKYSDTDADPHNATRGFFYSHVGWLLVRKHPEVKR

RGKELDMSDIYNNPVLRFQKKYAIPFIGAMCFGLPTFIPVYFWGETWSNAWHITMLRYILN

LNITFLVNSAAHIWGYKPYDIKILPAQNIAVSIVTGGEVSITTTTFFPWDYRAAELGNNYL

NLTTKFIDFFAWIGWAYDLKTVSSDVIKSKAERTGDGTNLWGLEDKGEEDFLKIWKDN

SEQ ID NO: 64

Amyelois transitella desaturase from DTU WO 2016/

207339_SEQ ID NO: 2

MVPNKGSSDVLSEHSEPQFTKLIAPQAGPRKYKIVYRNLLTFGYWHLSAVYGLYLCFTCAK

WATILFAFFLYVIAEIGITGGAHRLWAHRTYKAKLPLEILLLIMNSIAFQDTAFTWARDHR

LHHKYSDTDADPHNATRGFFYSHVGWLLVKKHPEVKARGKYLSLDDLKNNPLLKFQKKYAI

LVIGTLCFLMPTFVPVYFWGEGISTAWNINLLRYVMNLNMTFLVNSAAHIFGNKPYDKSIA

SVQNISVSLATFGEGFHNYHHTYPWDYRAAELGNNRLNMTTAFIDFFAWIGWAYDLKSVPQ

EAIAKRCAKTGDGTDMWGRKR

SEQ ID NO: 65

Spodoptera littoralis desaturase from DTU WO 2016/

207339_SEQ ID NO: 41

MAQCVQTTTILEQKEEKTVTLLVPQAGKRKFEIVYFNIITFAYWHIAGLYGLYLCFTSTKW

ATVLFSFFLFVVAEVGVTAGSHRLWSHKTYKAKLPLQILLMVMNSLAFQNTVIDWVRDHRL

HHKYSDTDADPHNASRGFFYSHVGWLLVRKHPDVKKRGKEIDISDIYNNPVLRFQKKYAIP

FIGAVCFVLPTLIPVYGWGETWTNAWHVAMLRYIMNLNVTFLVNSAAHIYGKRPYDKKILP

SQNIAVSIATFGEGFHNYHHVFPWDYRAAELGNNSLNFPTKFIDFFAWIGWAYDLK

TVSKEMIKQRSKRTGDGTNLWGLEDVDTPEDLKNTKGE

SEQ ID NO: 66

Agrotis segetum desaturase from DTU WO 2016/

207339_SEQ ID NO: 43

MAQGVQTTTILREEEPSLTFVVPQEPRKYQIVYPNLITFGYWHIAGLYGLYLCFTSAKWQT

ILFSFMLVVLAELGITAGAHRLWAHKTYKAKLPLQIILMILNSIAFQNSAIDWVRDHRLHH

KYSDTDADPHNATRGFFYSHVGWLLVRKHPEVKRRGKELDMSDIYNNPVLRFQKKYAIPFI

GAMCFGLPTFIPVYFWGETWSNAWHITMLRYILNLNITFLVNSAAHIWGYKPYDIKILPAQ

NIAVSIVTGGEVSITTTTFFPWDYRAAELGNNYLNLTTKFIDFFAWIGWAYDLKTVSSDVI

KSKAERTGDGTNLWGLEDKGEEDFLKIWKDN

SEQ ID NO: 67

Trichoplusia ni desaturase from DTU WO 2016/

207339_SEQ ID NO: 45

MAVMAQTVQETATVLEEEARTVTLVAPKTTPRKYKYIYTNFLTFSYAHLAALYGLYLCFTS

AKWETLLFSFVTFHMSNIGITAGAHRLWTHKTFKAKLPLEIVLMIFNSLAFQNTAITWARE

HRLHHKYSDTDADPHNASRGFFYSHVGWLLVKKHPDVLKYGKTIDMSDVYNNPVLKFQKKY

AVPLIGTVCFALPTLIPVYCWGESWNNAWHIALFRYIFNLNVTFLVNSAAHIWGNKPYDKS

ILPAQNLLVSFLASGEGFHNYHHVFPWDYRTAELGNNFLNLTTLFIDFCAWFGWAYDLKSV

SEDIIKQRAKRTGDGSSGVIWGWDDKDMDRDIKSKANIFYAKKE

SEQ ID NO: 68

Amyelois transitella desaturase from DTU WO 2016/

207339_SEQ ID NO: 1

atggttccaaacaagggttcctctgatgttttgtctgaacattctgaaccacaattcacca

agttgattgctccacaagctggtccaagaaagtacaaaatcgtttacagaaacttgttgac

cttcggttactggcatttgtctgctgtttatggtttgtacttgtgtttcacttgtgctaag

tgggctactattttgttcgctttcttcttgtacgttatcgccgaaattggtattactggtg

gtgctcatagattatgggctcatagaacttacaaagccaagttgccattggaaatcttgtt

gttgatcatgaactccattgccttccaagatactgcttttacttgggctagagatcataga

ttgcatcacaagtactctgatactgatgctgatccacataatgctactagaggtttcttct

actctcatgttggttggttgttggttaagaaacacccagaagttaaggctagaggtaagta

cttgtctttggatgacttgaagaacaaccctttgttgaagttccaaaagaagtacgccatt

ttggtcattggtactttgtgctttttgatgccaactttcgttccagtttacttttggggtg

aaggtatttctactgcctggaacattaacttgttaagatacgtcatgaacttgaacatgac

ctttttggttaactccgctgctcatatttttggtaacaagccatacgataagtctatcgcc

tctgttcaaaacatctctgtttctttggctactttcggtgaaggtttccataactaccatc

atacttatccatgggattacagagctgctgaattgggtaacaatagattga&tatgaccac

cgccttcattgatttctttgcttggattggttgggcctacgatttgaaatctgttccacaa

gaagctattgctaagagatgtgctaaaactggtgatggtactgatatgtggggtagaaaga

gatga

SEQ ID NO: 69

Spodoptera littoralis desaturase from DTU WO 2016/

207339_SEQ ID NO: 40

ggacactgacatggactgaaggagtagagaatcggcccgtggagttggccttcattttcag

tcttatctctcggtgttatggtagtcacttatatcggtattaaaataagtgaataaggctt

gtaaaaatggcgcaatgtgtacaaacaacaacgattttggaacaaaaagaagagaaaacag

taactttgctggtacctcaagcgggaaagaggaagtttgaaattgtgtattttaatatcat

caccttcgcttactggcatatagctggactatatggcctttatttgtgcttcacttcaaca

aaatgggcgacagttttattctcattctttctattcgtcgtagcagaagtaggggtcacgg

ctggctcccacagactttggtcgcataaaacttacaaagcaaaactacctttacaaattct

gctaatggtgatgaattcccttgcatttcaaaacacagtcattgattgggtgagagaccat

cgactccatcataagtatagcgacactgatgccgatccccataatgcctcccgaggatttt

tctattcgcacgtcggttggctgcttgtgagaaaacaccctgatgtcaagaaacgaggaaa

ggaaattgatatatctgatatttacaacaatccggtactgaggttccagaagaagtacgca

attcctttcatcggggcagtttgtttcgtcttaccaacattgataccggtttacggttggg

gagaaacctggactaatgcctggcacgtcgccatgctgcggtacattatgaaccttaacgt

caccttcctggtcaacagcgctgctcatatatatggaaagagaccttatgacaagaagatc

ctaccatctcaaaacatagctgtgtccattgcaacctttggggaaggtttccataattatc

atcatgtatttccatgggattatcgcgcagctgaacttggaaataacagtttgaatttccc

tacgaaatttattgatttctttgcgtggatcggatgggcgtatgacctaaagactgtttcg

aaagaaatgataaaacaaaggtcaaaaagaactggtgatggaactaatctatgggggttag

aagatgtggataccccggaggatttaaaaaatacaaaaggcgaataggcaaacccttaaac

tcaaacagtgaggtttaatgtgatatttagaattagaattaatttatttgaaattaaatga

aggttttggataactgtttttaataataaaaatagtttttcgattaaattccttagattat

tttaaaggaaatgtataaggtactcgcgtggttagcaacccagcagtccctgtttatctgt

ttttatgaatttattctatgaatgtagatgtcgcatgaaattttaaaatgttgcatttgta

taattttacttatgaataaataaatttatttttaaaaaaaaaaaaaaaaaaaaaaaaaaaa

aaaaaa

SEQ ID NO: 70

Agrotis segetum desaturase from DTU WO 2016/

207339_SEQ ID NO: 42

atggctcaaggtgtccaaacaactacgatattgagggaggaagagccgtcattgactttcg

tggtacctcaagaaccgagaaagtatcaaatcgtgtacccaaaccttatcacatttgggta

ctggcatatagctggtttatacgggctatatttgtgctttacttcggcaaaatggcaaaca

attttattcagtttcatgctcgttgtgttagcagagttgggaataacagccggcgctcaca

ggttatgggcccacaaaacatataaagcgaagcttcccttacaaatcatcctgatgatact

gaactccattgccttccaaaattccgccattgattgggtgagggaccaccgtctccatcat

aagtacagtgacactgatgcagaccctcacaatgctactcgtggtttcttctattctcatg

ttggatggttgctcgtaagaaaacatccagaagtcaagagacgtggaaaggaacttgacat

gtctgatatttacaacaatccagtgctgagatttcaaaagaagtatgctatacccttcatc

ggggcaatgtgcttcggattaccaacttttatccctgtttacttctggggagaaacctgga

gtaatgcttggcatatcaccatgcttcggtacatcctcaacctaaacattactttcctggt

caacagtgctgctcatatctggggatacaaaccttatgacatcaaaatattgcctgcccaa

aatatagcagtttccatagtaaccggcggcgaagtttccataactaccaccacgttttttc

cttgggattatcgtgcagcagaattggggaacaattatcttaatttgacgactaagttcat

agatttcttcgcttggatcggatgggcttacgatcttaagacggtgtccagtgatgttata

aaaagtaaggcggaaagaactggtgatgggacgaatctttggggtttagaagacaaaggtg

aagaagattttttgaaaatctggaaagacaattaa

SEQ ID NO: 71

Trichoplusia ni desaturase from DTU WO 2016/

207339_SEQ ID NO: 44

atggctgtgatggctcaaacagtacaagaaacggctacagtgttggaagaggaagctcgca

cagtgactcttgtggctccaaagacaacgccaaggaaatataaatatatatacaccaactt

tcttacattttcatatgcgcatttagctgcattatacggactttatttgtgcttcacctct

gcgaaatgggaaacattgctattctctttcgtactcttccacatgtcaaatataggcatca

ccgcaggggctcaccgactctggactcacaagactttcaaagccaaattgcctttggaaat

tgtcctcatgatattcsactctttagcctttcaaaacacggctattacatgggctagagaa

catcggctacatcacaaatacagcgatactgatgctgatccccacaatgcgtcaagagggt

tcttctactcgcatgttggctggctattagtaaaaaaacatcccgatgtcctgaaatatgg

aaaaactatagacatgtcggatgtatacaataatcctgtgttaaaatttcagaaaaagtac

gcagtacccttaattggaacagtttgttttgctcttccaactttgattccagtctactgtt

ggggcgaatcgtggaacaacgcttggcacatagccttatttcgatacatattcaatcttaa

cgtgactttcctagtcaacagtgctgcgcatatctgggggaataagccttatgataaaagc

atcttgcccgctcaaaacctgctggtttccttcctagcaagtggagaaggcttccataatt

accatcacgtctttccatgggattaccgcacagcagaattagggaataacttcctgaattt

gacgacgctgttcattgatttttgtgcctggtttggatgggcttatgacttgaagtctgta

tcagaggatattataaaacagagagctaaacgaacaggtgacggttcttcaggggtcattt

ggggatgggacgacaaagacatggaccgcgatataaaatctaaagctaacattttttatgc

taaaaaggaatga

SEQ ID NO: 72

Spodoptera exigua FAR-like protein VIII nucleotide sequence

(Genbank ID KR781121.1, codon optimized)

ATGGTGGTGCTGACCAGCAAGGAGAAGTCCAACATGTCTGTGGCCGACTTCTACGCCGGCA

AGTCCGTGTTTATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCT

GTACTCTTGCCCAGACATCGATAAGATCTATATGCTGATCCGGGAGAAGAAGGGCCAGAGC

ATCAGGGAGCGCCTGAGCAAGATCGTGGAGGATCCCCTGTTCAATAGGCTGAAGGAGAAGC

GCGCTGGCGACCTGGATAAGATCGTGCTGATCCCAGGCGATGTGACAGTGCCCGGCCTGGG

CATCTCCGACGAGAACGAGGCCATCCTGATCGATAAGGTGTCTGTGGTCATCCACAGCGCC

GCCACAGTGAAGTTCAATGAGCCCCTGGAGACCGCCTGGAACGTGAATGTGGAGGGCACAA

GGATGATCATGGCCCTGTCTCGGAAGATGAAGAGAATCGAGATCTTTATCCACATCAGCAC

CGCCTACACCAACACAAATAGGGCAGTGGTGGACGAGGTGCTGTACCCACCTCCAGCCGAC

ATCAACGAGGTGCACCAGTATGTGAAGAATGGCATCACAGAGGAGGAGACCGAGAAGATCC

TGAACGGCAGGCCCAATACCTACACATTCACCAAGGCCCTGACCGAGCACCTGGTGGCAGA

GAACCAGGCCTATATGCCTACAATCATCGTGCGGCCATCCATCGTGGGCGCCATCAAGGAC

GATCCTATCAGAGGCTGGCTGGCAAACTGGTACGGAGCAACAGGACTGAGCGTGTTCACCG

CCAAGGGCCTGAATCGCGTGATCTACGGCCAGAGCAGCCACGTGGTGGACCTGATCCCTGT

GGATTATGTGGCAAACCTGGTCATCGTGGCAGGAGCAAAGACATACCGGTCCAACGAGGTG

ACCATCTATAATTCTTGCTCTAGCTCCTGTAATCCAATCACAATGGAGCGGCTGGTGGGCC

TGTTCATCGACGATACAGTGAAGCACAACAGCTACGTGATGCCCCTGCCTGGCTGGTACGT

GTATTCCAATTACCGGTGGCTGGTGTATCTGGTGACCATCATCTTTCAGATGATCCCAGCC

TATCTGGCAGACATCGGCCGGAGACTGCTGGGCAAGAATCCCAGATACTATAAGCTGCAGT

CCCTGGTGGCACAGACCCAGGAGGCAGTGCACTTCTTTACATCTCACACCTGGGAGATCAA

GAGCAAGAGGACCTCCGAGCTGTTCGCCTCTCTGAGCCACACAGACCAGCGCATCTTTCCT

TGCGATGCCAAGAAGATCGACTGGACAGATTAGATCACCGATTATTGTAGCGGCGTGCGGC

AGTTCCTGGAGAAGAAGAAGTGA

SEQ ID NO: 73

Spodoptera exigua FAR-like protein VIII amino acid sequence

(Genbank ID ALJ94061.1)

MVVLTSKEKSNMSVADFYAGKSVFITGGTGFLGKVFIEKLLYSCPDIDKIYMLIREKKGQS

IRERLTKIVDDPLFNRLKEKRPGDLDKIVLIPGDVTVPGLGISDENEAILIDKVSVVIHSA

ATVKFNEPLETAWNVNVEGTRMIMALSRKMKRIEIFIHISTAYTNTNRAVVDEVLYPPPAD

INEVHQYVKNGITEEETEKILNGRPNTYTFTKALTEHLVAENQAYMPTIIVRPSIVGAIKD

DPIRGWLANWYGATGLSVFTAKGLNRVIYGQSSHVVDLIPVDYVANLVIVAGAKTYRSNEV

TIYNSCSSSCNPITMERLVGLFIDDTVKHNSYVMPLPGWYVYSNYRWLVYLVTIIFQMIPA

YLADIGRRLLGKNPRYYKLQSLVAQTQEAVHFFTSHTWEIKSKRTSELFASLSHTDQRIFP

CDAKKIDWTDYITDYCSGVRQFLEKKK

SEQ ID NO: 74

Euglena gracilis fatty acyl-coenzyme A reductase nucleotide

sequence, codon optimized

ATGAACGACTTCTACGCCGGCAAGGGCGTGTTTCTGACAGGCGTGACCGGCTTCGTGGGCA

AGATGGTGGTGGAGAAGATCCTGCGGTCTCTGCCAACCGTGGGCAGGCTGTATGTGCTGGT

GCGCCCAAAGGCAGGCACAGATCCTCACCAGAGACTGCACAGCGAAGTGTGGAGCAGCGCC

GGATTTGACGTGGTGAGGGAGAAAGTGGGAGGACCTGCAGCCTTCGATGCACTGATCCGCG

AGAAGGTGGTGCCTGTGCCAGGCGACATGGTGAAGGATAGGTTTGGCCTGGACGATGCAGC

ATACCGCTCCCTGGCAGCCAACGTGAATGTGATCATCCACATGGCCGCCACAATCGACTTC

ACCGAGAGGCTGGATGTGGCCGTGTCTCTGAACGTGCTGGGCACAGTGCGGGTGCTGACCC

TGGCAAGGAGAGCCAGAGAGCTGGGCGCCCTGCACAGCGTGGTGCACGTGTCCACCTGCTA

CGTGAACTCCAATCAGCCCCCTGGCGCCCGGCTGAGAGAGCAGCTGTATCCCCTGCCTTTT

GACCCACGGGAGATGTGCACAAGAATCCTGGACATGAGCCCTCGGGAGATCGATCTGTTCG

GCCCACAGCTGCTGAAGCAGTACGGCTTCCCCAATACCTATACCTTCACCAAGTGCATGGC

AGAGCAGCTGGGCGCCCAGATCGCACACGACCTGCCATTCGCCATCTTTAGACCAGCAATC

ATCGGAGCCGCCCTGTCCGAGCCATTTCCCGGCTGGTGCGATTCTGCCAGCGCCTGTGGAG

CCGTGTTCCTGGCAGTGGGACTGGGCGTGCTGCAGGAGCTGCAGGGAAACGCCTCTAGCGT

GTGCGACCTGATCCCTGTGGATCACGTGGTGAATATGCTGCTGGTGACAGCAGCATATACC

GCATCTGCCCCACCAGCCGACCCTAGCCCATCCTCTCTGGCCCTGTCCCCTCCACAGCTGC

CACTGGCCACACTGCCCCCTGGCACCGTGGCAGATGTGCCAATCTACCACTGTGGCACCTC

TGCCGGCCCTAACGCCGTGAATTGGGGCAGGATCAAGGTGAGCCTGGTGGAGTATTGGAAC

GCACACCCAATCGCAAAGACCAAGGCAGCAATCGCCCTGCTGCCCGTGTGGAGGTTCGAGC

TGAGCTTTCTGCTGAAGAGGCGCCTGCCTGCAACAGCCCTGTCCCTGGTGGCCTCTCTGCC

AGGCGCATCCGCCGCCGTGCGGAGACAGGCAGAGCAGACAGAGCGGCTGGTGGGCAAGATG

AGAAAGCTGGTGGACACCTTTCAGTCCTTCGTGTTTTGGGCCTGGTACTTCCAGACAGAGA

GCAGCGCCAGGCTGCTGGCCTCTCTGTGCCCAGAGGACCGCGAGACCTTTAACTGGGACCC

CAGGAGGATCGGATGGAGGGCCTGGGTGGAGAATTACTGTTATGGCCTGGTGCGGTATGTG

CTGAAGCAGCCAATCGGCGATAGACCACCAGTGGCAGCAGAGGAGCTGGCAAGCAATAGGT

TCCTGCGCGCCATGCTGTGA

SEQ ID NO: 75

Euglena gracilis fatty acyl-coenzyme A reductase amino acid

sequence (ADI60057.1)

MNDFYAGKGVFLTGVTGFVGKMVVEKILRSLPTVGRLYVLVRPKAGTDPHQRLHSEVWSSA

GFDVVREKVGGPAAFDALIREKVVPVPGDMVKDRFGLDDAAYRSLAANVNVIIHMAATIDF

TERLDVAVSLNVLGTVRVLTLARRARELGALHSVVHVSTCYVNSNQPPGARLREQLYPLPF

DPREMCTRILDMSPREIDLFGPQLLKQYGFPNTYTFTKCMAEQLGAQIAHDLPFAIFRPAI

IGAALSEPFPGWCDSASACGAVFLAVGLGVLQELQGNASSVCDLIPVDHVVNMLLVTAAYT

ASAPPADPSPSSLALSPPQLPLATLPPGTVADVPIYHCGTSAGPNAVNWGRIKVSLVEYWN

AHPIAKTKAAIALLPVWRFELSFLLKRRLPATALSLVASLPGASAAVRRQAEQTERLVGKM

RKLVDTFQSFVFWAWYFQTESSARLLASLCPEDRETFNWDPRRIGWRAWVENYCYGLVRYV

LKQPIGDRPPVAAEELASNRFLRAML

SEQ ID NO: 76

Yponomeuta evonymellus fatty-acyl CoA reductase II nucleotide

sequence, codon optimized

ATGGTGCAGCTGAAGGAGGACTCCGTGGCCGCCTTTTACGCCGAGAAGTCTATCTTCATCA

CAGGCGGCACCGGCTTTCTGGGCAAGGTGCTGATCGAGAAGCTGCTGTACTCCTGCAAGGC

CGTGGACCAGATCTATGTGCTGATCCGGAAGAAGAAGGATCAGACACCTTCTGAGCGCATC

GCCGAGCTGCTGGAGTCTGAGCTGTTCAGCCGGCTGAGAAAGGACGATCCAAGCGCCCTGA

AGAAGGTGGTGCCCGTGGTGGGCGACCTGACCATGCCTAACCTGGGACTGAGCGCCGCAGT

GCAGGATCTGATCGTGACAAAGGTGTCCATCATCTTCCACGTGGCCGCCACCGTGAAGTTT

AACGAGAGGATGAAGAATGCCCTGGCCAACAATGTGGAGGCCACCAGAGAAGTGATCAACC

TGTGCCACCGCCTGGAGAAGGTGGACGCCTTCATCCACGTGTCCACAGCCTATTCTAATAC

CGATCAGAAGGTGGTGGAGGAGCGCGTGTACCCACCTCCAGCACCTCTGAGCGAGGTGTAT

GCCTTTGTGACCAACAATGGCGACGATATGGACATCATCCAGAACCTGCTGAATGGCCGGC

CAAATACCTACACATATACGAAGGCCCTGGCCGAGGACATCGTGCTGAAGGAGCACGGCGG

CATCCCTACAGCCATCATCAGACGAAGCATCGTGCTGTCCGTGCTGAAGGAGCCCATCCCT

GGCTGGCTGGACAACTGGAATGGACCAACCGGACTGCTGCACGCCAGCTCCCAGGGAGTGC

ACTGCTCCATGCTGGGCTCTGGCAGCAACGTGGCCGACCTGATCCCTGTGGACATCGTGAC

AAATCTGATGATCGTGGTGGCCTCTCGGTGCAAGAAGAGCAACGGCCTGAAGGTGTACAAT

TCCTGTTCTGGCACCACAAACCCAATCGCCTATCAGGCCTTCACCAAGATGTTTCTGGATA

GCTGTATCTCCAGGGGCTGGAACAAGGTGCCATTCCCCATGCTGCTGTTTGTGAAGTGGGC

CTTCCTGAATCGCGTGCTGAAGTTCTTCCTGGTCATCGTGCCATTCTTTCTGATCGACGTG

TACCTGCGGTTCTTTGGCAAGCCCAATTACATGAGAATGATCACATATACCAAGAAGGCCG

AGGATCTGATGACATTCTTTACCTCTCACGAGTGGCAGTTCAAGGACGGCAACGTGCGGGA

TCTGATCAATATGATGAGCCCCGAGGATAGAAAGATCTTTTACTGCGACCCCGATGAGATC

CACTGGAAGCCTTACTTCGACGATTATTGCGTGGGCGTGTTTAAGTATCTGCTGAAGAGGA

AGGTGTGA

SEQ ID NO: 77

Yponomeuta evonymellus fatty-acyl CoA reductase II amino acid

sequence (ADD62439.1)

MVQLKEDSVAAFYAEKSIFITGGTGFLGKVLIEKLLYSCKAVDQIYVLIRKKKDQTPSERI

AQLLESELFSRLRKDDPSALKKVVPVVGDLTMPNLGLSAAVQDLIVTKVSIIFHVAATVKF

NERMKNALANNVEATREVINLCHRLEKVDAFIHVSTAYSNTDQKVVEERVYPPPAPLSEVY

AFVTNNGDDMDIIQNLLNGRPNTYTYTKALAEDIVLKEHGGIPTAIIRPSIVLSVLKEPIP

GWLDNWNGPTGLLHASSQGVHCSMLGSGSNVADLIPVDIVTNLMIVVASRCKKSNGLKVYN

SCSGTTNPIAYQAFTKMFLDSCISRGWNKVPFPMLLFVKWAFLNRVLKFFLVIVPFFLIDV

YLRFFGKPNYMRMITYTKKAEDLMTFFTSHEWQFKDGNVRDLINMMSPEDRKIFYCDPDEI

HWKPYFDDYCVGVFKYLLKRKV

SEQ ID NO: 78

Drosophila melanogaster fatty acid desaturase (Q9N9Z8)

mapysriyhqdkssretgvlfeddaqtvdsdlttdrfqlkraekrrlplvwrniilfalvh

laalyglhsiftraklattlfaaglyiigmlgvtagahrlwahrtykakwplrlllvifnt

iafqdavyhwardhrvhhkysetdadphnatrgfffshvgwllckkhpdikekgrgldlsd

lradpilmfqrkhyyilmplacfvlptvipmvywnetlasswfvatmfrwcfqlnmtwlvn

saahkfgnrpydktmnptqnafvsaftfgegwhnyhhafpwdyktaewgcyslnittafid

lfakigwaydlktvapdviqrrvlrtgdgshelwgwgdkdltaedarnvllvdksr

SEQ ID NO: 79

Lampronia capitella acyl-CoA-delta 11-desaturase (ABX71630.1)

mppypeevdtnhifeedisheeskpalkplvapqadnrkpeivplnlitfgyghlaaiygi

ylcftsakwativfafvlyicaelgitagahrlwshrsykaklplrlilllfntlafqnta

idwvrdhrmhhkysdtdadphnatrgfffshvgwlltrkhpevkrrgkdidmmdiyndsll

kfqkkyaipfvglvcfviptlmpmyfwnetlnnswhiatmlryivnlnmtflvnsaahiwg

ykpydksikpvqnitvsililgegfhnyhhvfpwdyrtselgndflnfttlfinlfakigw

aydlktasdkvvaarrkrtgdgtnlwgwedkslneeerqaatvlypnkylnlkd

SEQ ID NO: 80

Cydia pomonella desaturase (AIM40221.1)

mapnvtdvngvlfesdaatpdlalanapvqqaddspriyvwrniilfaylhiaalyggylf

lvsakwqtdifayflyvasglgitagahrlwahksykakwplrlilvifntiafqdsaidw

ardhrmhhkysetdadphnatrgfffshigwllvrkhpelkrkgkgldlsdlyadpilrfq

kkyylilmplacfvlptvipvylwnetwtnaffvaalfryafilnvtwlvnsaahkwgdkp

ydksikpsenisvslfafgegfhnyhhtfpwdyktaelssnrlnfttkfinffakigwayd

mktvsdeiiqkrvnrtgdgshhlwgwgdkdhskeevnaavrinpkdd

SEQ ID NO: 81

Spodoptera exigua FAR-like protein VII nucleotide

sequence(KF805977.1), codon optimized

ATGACGTATAGACAAATAAATGAATTTGATGCTGAAAAGTTTACGGCAGCTACAGTACCGA

CAAGCTACGTATCAGTACCAGATTTTTATGCGGGCAAGACAATTTTTATCACTGGTGGAAC

TGGATTTCTTGGAAAGGTGTTTCTAGAGAAACTTCTTTACAGTTGTAAAGATGTTGAAACC

GTATACATTTTGATCAGAGAGAAAAAAGGCAAAACACCTCAGCAAAGAGTTGAAGATCTTT

TTAACAAACCGATTTTCTCAAGATTGAAACAGAAGGACTCTCAGTGTATGAAGAAAGTCAC

TGCAATAATTGGTGACCTTAGTGAACCTGGTCTTGGCATATCAAAAGATGATGAAGAACTA

CTTTTGCAAAAGGTATCTGTAGTATTCCATGTCGCAGCCAATGTTCAGTTTTACAAGGAAT

TCAAAGAGATTATAAATACGAATGTTGGTGGGACAAAATACGTACTCCAATTGTGTCAGCG

AATAAAAGATATTAAGGCATTTGTCCATATTTCCACAGCCTACTGTCACACAGACCAAAAG

GTATTAGAAGAGAGAATATACCCCCCTCCAGCAGAACTCAGTGAAGTCCTGAAGTTCCTTC

AGCAGCCACAGCATGACAAGAAACAGATTAAGGAATTATTTAAGAAACAACCAAACAGTTA

CACCTTTGCCAAGGCTTTAGCAGAAACCTACATTGCTGAGAACTGCGGACGCGTCCCCACA

ATTATCATCAGACCTTCTATTATATCAGCATCACTGAAAGAGCCGCTACCAGGATGGGTGG

ATTCATGGAACGGAGCCACAGGCCTCATCACAGCTAGCTACAACGGCGCCAACAGAGTGCT

TCTCGGCGAAGGCAGCAACTTCCTCGACCTGATCCCAGTTGACTTTGTTGCTAACCTGGCA

ATTGTAGCTGCTGCTAAATGTACTAGCTCTTTGAAAGTTTACAATTGCTGCTCAAGCGGAT

GTAACCCTTTAACATTGAAACAATTGGTCAGCCACATGAATAATGTCGGATTTGATAAAAA

CGTCTCCATAATATTCACCAATAACAAAGCCTCGCTTTCCACATTGACATTTTTCCTTCAA

ACAACGCCATCTTTCACCGCTGATATGTTTCTGAGAGTCACGGGAAAGTCACCAAGGTACA

TGAAAATCCAGTCAAAACTGACCATCGCTCGGAATGCCTTAAATTTTTTCACCTGTCATTC

CTGGGTCATGAAGGCTGATAATTCTAGAAGACTGTATGCTTCCTTGTCATTACACGACCGA

CATACGTTCCCTTGTGATCCTACAGACATAGACTGGAAGAAGTACATAAATATATACATAG

AAGGAATTAATCAGTTCTTAATGAAGAAACGTAGTTAA

SEQ ID NO: 82

Spodoptera exigua FAR-like protein VII amino acid

sequence (AIS85928.1)

MTYRQINEFDAEKFTAATVPTSYVSVPDFYAGKTIFITGGTGFLGKVFLEKLLYSCKDVET

VYILIREKKGKTPQQRVEDLFNKPIFSRLKQKDSQCMKKVTAIIGDLSEPGLGISKDDEEL

LLQKVSVVFHVAANVQFYKEFKEIINTNVGGTKYVLQLCQRIKDIKAFVHISTAYCHTDQK

VLEERIYPPPAELSEVLKFLQQPQHDKKQIKELFKKQPNSYTFAKALAETYIAENCGRVPT

IIIRPSIISASLKEPLPGWVDSWNGATGLITASYNGANRVLLGEGSNFLDLIPVDFVANLA

IVAAAKCTSSLKVYNCCSSGCNPLTLKQLVSHMNNVGFDKNVSIIFTNNKASLSTLTFFLQ

TTPSFTADMFLRVTGKSPRYMKIQSKLTIARNALNFFTCHSWVMKADNSRRLYASLSLHDR

HTFPCDPTDIDWKKYINIYIEGINQFLMKKRS

SEQ ID NO: 83
HaFAR S60A FAR2

ATGGTGGTGCTGACCTCCAAGGAGACAAAGCCCTCTGTGGCCGAGTTCTACGCCGGCAAGA

GCGTGTTCATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTA

CAGCTGCCCTGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGGCCTGGCCGTG

TCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACAAGGCTGAAGGACAAGCGCC

CTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAGATCTGGGCAT

CAACAGCGAGAATGAGAAGATGCTGATCGAGAAGGTGAGCGTGATCATCCACTCCGCCGCC

ACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGA

TGATGCTGGCCCTGTCTCGGAGAATGAAGCGCATCGAGGTGTTTATCCACATGAGCACAGC

CTACACCAACACAAATAGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATC

GATGAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAGAAGATCCTGA

ACGGCAGACCCAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGGTGGCCGAGAA

CCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAG

CCTCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCCA

AGGGCCTGAATAGAGTGATCTACGGCCACACCTCCTATATCGTGGACCTGATCCCAGTGGA

TTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGTCTAGCAAGTCCACCGAGCTGAAG

GTGTATAACTGCTGTTCCTCTAGCTGTAATCCCGTGACCATCGGCACACTGATGAGCATGT

TCGCCGACGATGCCATCAAGCAGAAGTCCTACGCCATGCCTCTGCCAGGCTGGTACATCTT

TACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCATCCCTGCCTAC

GTGACCGACCTGTCTAGGCACCTGATCGGCAAGAGCCCACGCTATATCAAGCTGCAGAGCC

TGGTGAACCAGACCAGGTCCTCTATCGACTTCTTTACAAATCACTCCTGGGTCATGAAGGC

CGATAGGGTGCGCGAGCTGTACGCATCTCTGAGCCCAGCCGACAAGTATCTGTTCCCTTGC

GACCCAACCGATATCAACTGGACACACTACATCCAGGATTATTGTTGGGGCGTGCGCCACT

TTCTGGAGAAGAAGTCCTATGAGTGA

SEQ ID NO: 84
HaFAR S195A FAR3

ATGGTGGTGCTGACCTCCAAGGAGACAAAGCCCTCTGTGGCCGAGTTCTACGCCGGCAAGA

GCGTGTTCATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTA

CAGCTGCCCTGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGGCCTGAGCGTG

TCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACAAGGCTGAAGGACAAGCGCC

CTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAGATCTGGGCAT

CAACAGCGAGAATGAGAAGATGCTGATCGAGAAGGTGAGCGTGATCATCCACTCCGCCGCC

ACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGA

TGATGCTGGCCCTGTCTCGGAGAATGAAGCGCATCGAGGTGTTTATCCACATCAGCACAGC

CTACACCAACACAAATAGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATC

GATCAGGTGCACCAGTATGTGAAGGACGGCATCGCCGAGGAGGATACCGAGAAGATCCTGA

ACGGCAGACCCAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGGTGGCCGAGAA

CCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAG

CCTCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCCA

AGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTGATCCCAGTGGA

TTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGTCTAGCAAGTCCACCGAGCTGAAG

GTGTATAACTGCTGTTCCTCTAGCTGTAATCCCGTGACCATCGGCACACTGATGAGCATGT

TCGCCGACGATGCCATCAAGCAGAAGTCCTACGCCATGCCTCTGCCAGGCTGGTACATCTT

TACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCATCCCTGCCTAC

GTGACCGACCTGTCTAGGCACCTGATCGGCAAGAGCCCACGCTATATCAAGCTGCAGAGCC

TGGTGAACCAGACCAGGTCCTCTATCGACTTCTTTACAAATCACTCCTGGGTCATGAAGGC

CGATAGGGTGCGCGAGCTGTACGCATCTCTGAGCCCAGCCGACAAGTATCTGTTCCCTTGC

GACCCAACCGATATCAACTGGACAGACTACATCCAGGATTATTGTTGGGGCGTGCGCCACT

TTCTGGAGAAGAAGTCCTATGAGTGA

SEQ ID NO: 85
HaFAR S298A FAR4

ATGGTGGTGCTGACCTCCAAGGAGACAAAGCCCTCTGTGGCCGAGTTCTACGCCGGCAAGA

GCGTGTTCATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTA

CAGCTGCCCTGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGGCCTGAGCGTG

TCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACAAGGCTGAAGGACAAGCGCC

CTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGGACCAGATCTGGGCAT

CAACAGCGAGAATGAGAAGATGCTGATCGAGAAGGTGAGCGTGATCATCCACTCCGCCGCC

ACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGA

TGATGCTGGCCCTGTCTCGGAGAATGAAGCGCATCGAGGTGTTTATCCACATCAGCACAGC

CTACACCAACACAAATAGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATC

GATCAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAGAAGATCCTGA

ACGGCAGACCCAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGGTGGCCGAGAA

CCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAG

CCTCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCCA

AGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTGATCCCAGTGGA

TTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGGCCAGCAAGTCCACCGAGCTGAAG

GTGTATAACTGCTGTTCCTCTAGCTGTAATCCCGTGACCATCGGCACACTGATGAGCATGT

TCGCCGACGATGCCATCAAGCAGAAGTCCTAGGCCATGCCTCTGCCAGGCTGGTACATCTT

TACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCATCCCTGCCTAC

GTGACCGACCTGTCTAGGCACCTGATCGGCAAGAGCCCACGCTATATCAAGCTGCAGAGCC

TGGTGAACCAGACCAGGTCCTCTATCGACTTCTTTACAAATCACTCCTGGGTCATGAAGGC

CGATAGGGTGCGCGAGCTGTACGCATCTCTGAGCCCAGCCGACAAGTATCTGTTCCCTTGC

GACCCAACCGATATCAACTGGACACACTACATCCAGGATTATTGTTGGGGCGTGCGCCACT

TTCTGGAGAAGAAGTCCTATGAGTGA

SEQ ID NO: 86
HaFAR S378A FAR5

ATGGTGGTGCTGACCTCGAAGGAGAGAAAGCCCTCTGTGGCCGAGTTCTACGCCGGCAAGA

GCGTGTTCATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTA

CAGCTGCCCTGACATCGAGAAGATCTATATGCTGATCCGGGAGAAGAAGGGCCTGAGCGTG

TCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACAAGGCTGAAGGACAAGCGCC

CTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAGATCTGGGCAT

CAACAGCGAGAATGAGAAGATGCTGATCGAGAAGGTGAGCGTGATCATCCACTCCGCCGCC

ACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGA

TGATGCTGGCCCTGTCTCGGAGAATGAAGCGCATCGAGGTGTTTATCCACATCAGCACAGC

CTACACCAACACAAATAGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATC

GATCAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAGAAGATCCTGA

ACGGCAGACCCAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGGTGGCCGAGAA

CCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAG

CCTCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCCA

AGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTGATCCCAGTGGA

TTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGTCTAGCAAGTCCACCGAGCTGAAG

GTGTATAACTGCTGTTCCTCTAGCTGTAATCCCGTGACCATCGGCACACTGATGAGCATGT

TCGCCGACGATGCCATCAAGCAGAAGTCCTACGCCATGCCTCTGCCAGGCTGGTACATCTT

TACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCATCCCTGCCTAC

GTGACCGACCTGTCTAGGCACCTGATCGGCAAGGCCCGACGCTATATCAAGCTGCAGAGCC

TGGTGAACCAGACCAGGTCCTCTATCGACTTCTTTACAAATCACTCCTGGGTCATGAAGGC

CGATAGGGTGCGCGAGCTGTACGCATCTCTGAGCCCAGCCGACAAGTATCTGTTCCCTTGC

GACCCAACCGATATCAACTGGACACACTACATCCAGGATTATTGTTGGGGCGTGCGCCACT

TTCTGGAGAAGAAGTCCTATGAGTGA

SEQ ID NO: 87
HaFAR S394A FAR6

ATGGTGGTGCTGACCTCCAAGGAGACAAAGCCCTCTGTGGCCGAGTTCTACGCCGGCAAGA

GCGTGTTCATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTA

CAGCTGCCCTGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGGCCTGAGCGTG

TCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACAAGGCTGAAGGACAAGCGCC

CTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAGATCTGGGCAT

CAACAGCGAGAATGAGAAGATGCTGATCGAGAAGGTGAGCGTGATCATCCACTCCGCCGCC

ACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGA

TGATGCTGGCCCTGTCTCGGAGAATGAAGCGCATCGAGGTGTTTATCCACATCAGCACAGC

CTACACCAACACAAATAGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATC

GATCAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAGAAGATCCTGA

ACGGCAGACCCAATACCTACACATTCACCAAGGCCCTGACAGAGCAGCTGGTGGCCGAGAA

CCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAG

CCTCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCCA

AGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTCATCCGAGTGGA

TTACGTGGCAAACCTGGTCATCGCAGCAGGAGCGAAGTCTAGCAAGTCCACCGAGCTGAAG

GTGTATAACTGCTGTTCCTCTAGCTGTAATCCCGTGACCATCGGCACACTGATGAGGATGT

TCGCCGACGATGCCATCAAGCAGAAGTCCTACGCCATGCCTCTGCCAGGCTGGTACATCTT

TACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCATCCCTGCCTAC

GTGACCGACCTGTCTAGGCACCTGATCGGCAAGAGCCCACGCTATATCAAGCTGCAGAGCC

TGGTGAACCAGACCAGGTCCGCCATCGACTTCTTTACAAATCACTCCTGGGTGATGAAGGC

CGATAGGGTGCGCGAGCTGTACGCATCTCTGAGCCCAGCCGACAAGTATCTGTTCCCTTGC

GACCCAACCGATATCAACTGGACACACTACATCCAGGATTATTGTTGGGGCGTGCGCCACT

TTCTGGAGAAGAAGTCCTATGAGTGA

SEQ ID NO: 88
HaFAR S418A FAR7

ATGGTGGTGCTGACCTCCAAGGAGACAAAGCCCTCTGTGGCCGAGTTCTACGCCGGCAAGA

GCGTGTTCATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTA

CAGCTGCCCTGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGGCCTGAGCGTG

TCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACAAGGCTGAAGGACAAGCGCC

CTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAGATCTGGGCAT

CAACAGCGAGAATGAGAAGATGCTGATCGAGAAGGTGAGCGTGATCATCCACTCCGCCGCC

ACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGA

TGATGCTGGCCCTGTCTCGGAGAATGAAGCGCATCGAGGTGTTTATCCACATCAGCACAGC

CTACACCAACACAAATAGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATC

GATCAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAGAAGATCCTGA

ACGGCAGACCCAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGGTGGCCGAGAA

CCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAG

CCTCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCCA

AGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTGATCCCAGTGGA

TTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGTCTAGCAAGTCCACCGAGCTGAAG

GTGTATAACTGCTGTTCCTCTAGCTGTAATCCCGTGACCATCGGCACACTGATGAGCATGT

TCGCCGACGATGCCATCAAGCAGAAGTCCTACGCCATGCCTCTGCCAGGCTGGTACATCTT

TACAAAGTATAAGTGGCTGGTGCTGCTGCTCACCTTCCTGTTTCAGGTCATCCCTGCCTAC

GTGACCGACCTGTCTAGGCACCTGATCGGCAAGAGCCCACGCTATATCAAGCTGCAGAGCC

TGGTGAACCAGACCAGGTCCTCTATCGACTTCTTTACAAATCACTCCTGGGTCATGAAGGC

CGATAGGGTGCGCGAGCTGTACGCATCTCTGGCCCCAGCCGACAAGTATCTGTTCCCTTGC

GACCGAACCGATATCAACTGGACACACTACATCCAGGATTATTGTTGGGGCGTGCGCCACT

TTCTGGAGAAGAAGTCCTATGAGTGA

SEQ ID NO: 89
HaFAR S453A FAR8

ATGGTGGTGCTGACCTCCAAGGAGACAAAGCCCTCTGTGGCCGAGTTCTACGCCGGCAAGA

GCGTGTTCATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTA

CAGCTGCCCTGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGGCCTGAGCGTG

TCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACAAGGCTGAAGGACAAGCGCC

CTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAGATCTGGGCAT

CAACAGCGAGAATGAGAAGATGCTGATCGAGAAGGTGAGCGTGATCATCCACTCCGCCGCC

ACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGA

TGATGCTGGCCCTGTCTCGGAGAATGAAGCGCATCGAGGTGTTTATCCACATCAGCACAGC

CTACACCAACACAAATAGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATC

GATCAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAGAAGATCCTGA

ACGGCAGACCCAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGGTGGCCGAGAA

CCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAG

CCTCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCGA

AGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTGATCCCAGTGGA

TTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGTCTAGGAAGTCCACCGAGCTGAAG

GTGTATAACTGCTGTTCCTCTAGCTGTAATCCCGTGACCATCGGCACACTGATGAGCATGT

TCGCCGACGATGCGATCAAGCAGAAGTCCTACGCCATGCCTCTGCCAGGCTGGTACATCTT

TACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCATCCCTGCCTAC

GTGACCGACCTGTCTAGGCACCTGATCGGCAAGAGCCCACGCTATATCAAGCTGCAGAGCC

TGGTGAACCAGACCAGGTCCTCTATCGACTTCTTTACAAATCACTCCTGGGTCATGAAGGC

CGATAGGGTGCGCGAGCTGTACGCATCTCTGAGCCCAGCCGACAAGTATCTGTTCCCTTGC

GACCCAACCGATATCAACTGGACACACTACATCCAGGATTATTGTTGGGGCGTGCGCCACT

TTCTGGAGAAGAAGGCCTATGAGTGA

SEQ ID NO: 90
Codon optimized wild type HaFAR (FAR9; Strain SPV916)

ATGGTGGTGCTGACCTCGAAGGAGACAAAGCCCTCTGTGGCCGAGTTCTACGCCGGCAAGA

GCGTGTTCATCACAGGCGGCACCGGCTTCCTGGGCAAGGTGTTTATCGAGAAGCTGCTGTA

CAGCTGCCCTGACATCGAGAACATCTATATGCTGATCCGGGAGAAGAAGGGCCTGAGCGTG

TCCGAGAGAATCAAGCAGTTCCTGGACGATCCCCTGTTTACAAGGCTGAAGGACAAGCGCC

CTGCCGATCTGGAGAAGATCGTGCTGATCCCAGGCGACATCACCGCACCAGATCTGGGCAT

CAACAGCGAGAATGAGAAGATGCTGATCGAGAAGGTGAGCGTGATCATCCACTCCGCCGCC

ACCGTGAAGTTCAACGAGCCCCTGCCTACAGCCTGGAAGATCAATGTGGAGGGCACCAGGA

TGATGCTGGCCCTGTCTCGGAGAATGAAGCGCATCGAGGTGTTTATCCACATCAGCACAGC

CTACACCAACACAAATAGGGAGGTGGTGGACGAGATCCTGTACCCAGCCCCCGCCGACATC

GATCAGGTGCACCAGTATGTGAAGGACGGCATCAGCGAGGAGGATACCGAGAAGATCCTGA

ACGGCAGACCCAATACCTACACATTCACCAAGGCCCTGACAGAGCACCTGGTGGCCGAGAA

CCAGGCCTATGTGCCTACCATCATCGTGAGACCATCCGTGGTGGCCGCCATCAAGGATGAG

CCTCTGAAGGGATGGCTGGGAAACTGGTTCGGAGCAACAGGACTGACCGTGTTTACAGCCA

AGGGCCTGAATAGAGTGATCTACGGCCACAGCTCCTATATCGTGGACCTGATCCCAGTGGA

TTACGTGGCAAACCTGGTCATCGCAGCAGGAGCCAAGTCTAGCAAGTCCACCGAGCTGAAG

GTGTATAACTGCTGTTCCTCTAGCTGTAATCCCGTGACCATCGGCACACTGATGAGCATGT

TCGCCGACGATGCCATCAAGCAGAAGTCCTACGCCATGCCTCTGCCAGGCTGGTACATCTT

TACAAAGTATAAGTGGCTGGTGCTGCTGCTGACCTTCCTGTTTCAGGTCATCCCTGCCTAC

GTGACCGACCTGTCTAGGCACCTGATCGGCAAGAGCCCACGCTATATCAAGCTGCAGAGCC

TGGTGAACCAGACCAGGTCCTCTATCGACTTCTTTACAAATCACTCCTGGGTCATGAAGGC

CGATAGGGTGCGCGAGCTGTACGCATCTCTGAGCCCAGCCGACAAGTATCTGTTCCCTTGC

GACCCAACCGATATCAACTGGACACACTACATCCAGGATTATTGTTGGGGCGTGCGCCACT

TTCTGGAGAAGAAGTCCTATGAGTGA

SEQ ID NO: 91
PdDGAT1A (Phoenix dactylifera DGAT1A)

ATGGCCATCCCATCCGATAGAGAGACCCTGGAGAGGGCACCAGAGCCTTCTCCAGCAAGCG

ACCTGCAGAGCTCCCTGCGGAGAAGGCTGCACTCTACCGTGGCAGCAGTGGTGGTGCCAGA

TTCTAGCTCCAAGACATCTAGCCCCAGCGCCGAGAACCTGACCACAGACAGCGGAGAGGAT

TCCAGGGGCGACACCTCCTCTGACGCCGATACAAGGGATAGGGTGGTGGACGGAGTGGATA

GGGAGGAGGAGAACAAGACCGTGAGCGTGCTGAATGGCAGACAGTACGAGGACGGAGGCGG

CAGGGGACAGGGACAGGGCACAGGCGGCGGCGTGCCCGCCAAGTTTCTGTATAGGGCATCT

GCCCCTGCACACAGGAAGGTGAAGGAGAGCCCACTGAGCTCCGATGCCATCTTCAAGCAGA

GCCACGCCGGCCTGCTGAACCTGTGCATCGTGGTGCTGATCGCCGTGAACTCCAGGCTGAT

CATCGAGAATCTGATGAAGTACGGCCTGCTGATCCGCGCCGGCTATTGGTTTTCTAGCAAG

TCCCTGCGGGACTGGCCTCTGCTGATGTGCTGTCTGACCCTGCCAGCATTTCCTCTGGGAG

CCTTCATGGTGGAGAAGCTGGCCCAGCACAATTTGATCTCCGAGTCTGTGGTCATGAGCCT

GCACGTGATCATCACCACAGCCGAGCTGCTGTACCCAGTGATCGTGATCCTGAGATGCGAT

TCTGCCGTGCTGAGCGGCATCACACTGATGCTGTTTGCCAGCGTGGTGTGGCTGAAGCTGG

TGTCCTACGCCCACACCAACTATGACATGAGGACACTGAGCAAGTCCATCGACAAGGAGGA

TATGTACTCCAAGTGTCCAGAGATCGATAATCTGAAGGGCGACTCCTTTAAGTCTCTGGTG

TATTTCATGGTGGCCCCCACCCTGTGCTACCAGCCAAGCTATCCAAGGACCACCTGCATCA

GGAAGGGATGGGTCATCCGCCAGGTGGTGAAGCTGGTCATCTTCACCGGCCTGATGGGCTT

CATCATCGAGCAGTACATCAACCCCATCGTGCAGAATTCCCAGCACCCTCTGAAGGGCAAC

TTTCTGAATGCCATCGAGCGGGTGCTGAAGCTGTCTGTGCCCACCCTGTACGTGTGGCTGT

GCATGTTCTATTGTTTCTTTCACCTGTGGCTGAACATCCTGGCCGAGCTGCTGTGCTTTGG

CGATAGAGAGTTCTACAAGGACTGGTGGAACGCCAAGACAATCGAGGAGTATTGGAGGATG

TGGAATATGCCTGTGCACCGCTGGATGATCCGGCACATCTACTTCCCTTGTCTGAGAAATG

GCCTGCCAAGGGCCGTGGCCATCCTGATCTCCTTTCTGGTGTCTGCCATCTTCCACGAGAT

CTGCATCGCCGTGCCCTGTCACATCTTTAAGTTCTGGGCCTTTATCGGCATCATGTTCCAG

ATCCCCCTGGTCATCCTGACCAAGTATCTGCAGCACAAGTTTACAAACTCCATGGTGGGCA

ATATGATCTTCTGGTTCTTTTTCTCTATCCTGGGCCAGCCTATGTGCGTGCTGCTGTACTA

TCACGACGTGATGAATAGAAAGGTGAGGACCGAGTGA

SEQ ID NO: 92
PdDGAT1A (Phoenix dactylifera DGAT1A) protein encoded by SEQ

ID NO 91

MAIPSDRETLERAPEPSPASDLQSSLRRRLHSTVAAVVVPDSSSKTSSPSAENLTTDSGED

SRGDTSSDADTRDRVVDGVDREEENKTVSVLNGRQYEDGGGRGQGQGTGGGVPAKFLYRAS

APAHRKVKESPLSSDAIFKQSHAGLLNLCIVVLIAVNSRLIIENLMKYGLLIRAGYWFSSK

SLRDWPLLMCCLTLPAFPLGAFMVEKLAQHNFISESVVISLHVIITTAELLYPVIVILRCD

SAVLSGITLMLFASVVWLKLVSYAHTNYDMRTLSKSIDKEDMYSKCPEIDNLKGDSFKSLV

YFMVAPTLCYQPSYPRTTCIRKGWVIRQVVKLVIFTGLMGFIIEQYINPIVQNSQHPLKGN

FLNAIERVLKLSVPTLYVWLCMFYCFFHLWLNILAELLCFGDREFYKDWWNAKTIEEYWRM

WNMPVHRWMIRHIYFPCLRNGLPRAVAILISFLVSAIFHEICIAVPCHIFKFWAFIGIMFQ

IPLVILTKYLQHKFTNSMVGNMIFWFFFSILGQPMCVLLYYHDVMNRKVRTE

SEQ ID NO: 93
TEF Promoter for enzyme expression

GAGACCGGGTTGGCGGCGCATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCC

CAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCTCCCCACATATCAAAC

CTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCT

TGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAA

ATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACC

ACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAA

TCGTTAAGCATTTCCTTCTGAGTATAAGAATGATTCAAA

SEQ ID NO: 94
DST076_coding_sequence Z9 Desaturase

ATGCACATCGAGTCTGAGAACTGCCCCGGCAGGTTTAAGGAGGTGAACATGGCCCCTAATG

CCACCGATGCCAATGGCGTGCTGTTCGAGACCGATGCCGCCACACCTGACCTGGCCCTGCC

ACACGCACCTGTGCAGCAGGCCGAGAACTAGCCAAAGAAGTACGTGTGGCGCAATATCATC

CTGTTTGCCTACCTGCACATCGCCGCCCTGTACGGCGGCTATCTGTTTCTGTTCCACGCCA

AGTGGGAGACCGATATCTTGGCCTACATCCTGTATGTGATGTCTGGAGTGGGAATGACAGC

AGGAGCACACAGGCTGTGGGCCCACAAGAGCTACAAGGCCAAGTGGCCTCTGAGACTGATC

CTGGTCATCTTCAACACACTGGCCTTTCAGGACTCTGCCATCGATTGGAGCAGGGACCACC

GCATGCACCACAAGTATTCCGAGAGCGACGCCGATCCCCACAATGGCACACGGGGCTTCTT

TTTCTCTCACATCGGCTGGCTGCTGGTGCGGAAGCACCCTGAGCTGAAGAGAAAGGGCAAG

GGCCTGGACCTGTGCGATCTGTATGCCGACCCAATCCTGAGATTTCAGAAGAAGTACTATC

TGATCCTGATGCCCCTGACCTGTTTCGTGCTGCCAACAGTGATCCCCGTGTACTATTGGGG

CGAGACCTGGACAAACGCCTTTTTCGTGGCCGCCCTGTTTAGGTACGCCTTCATCCTGAAC

GTGACCTGGCTGGTGAATAGCGCCGCCCACAAGTGGGGGGATAAGGCTTATGAGCGCAACA

TCAAGCCATCCGAGAATATCAGCGTGTCCATGTTTGCCCTGGGCGAGGGCTTCCACAACTA

CCACCACACCTTCCCATGGGATTATAAGACAGCCGAGCTGGGCAACAATATGCTGAACTTC

ACCACAAACTTCATCAACTTCTTCGCCAAGATCGGCTGGGCCTACGATCTGAAGACCGTGT

CCGACGAGATCGTGCGGTCTACAGCAAAGAGGACAGGCGACGGAAGCCACCACCTGTGGGG

ATGGGGCGACAAGGATCACTCCAGGGAGGAGATGGCTGCCGCCATGCGCATCGACCCCAAG

GACGATTGA

SEQ ID NO: 95
DST076_amino_acid Z9 Desaturase encoded by SEQ ID NO 94

MHIESENCPGRFKEVNMAPNATDANGVLFETDAATPDLALPHAPVQQADNYPKKYVWRNII

LFAYLHIAALYGGYLFLFHAKWQTDIFAYILYVMSGLGITAGAHRLWAHKSYKAKWPLRLI

LVIFNTLAFQDSAIDWSRDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPELKRKGK

GLDLSDLYADPILRFQKKYYLILMPLTCFVLPTVIPVYYWGETWTNAFFVAALFRYAFILN

VTWLVNSAAHKWGDKPYDRNIKPSENISVSMFALGEGFHNYHHTFPWDYKTAELGNNMLNF

TTNFINFFAKIGWAYDLKTVSDEIVRSRAKRTGDGSHHLWGWGDKDHSREEMAAAIRIHPK

DD

SEQ ID NO: 96
DST180_coding_sequence Z9 Desaturase

ATGGCCCCAAACATCTCTGACGATGTGAATGGCGTGCTGTTTGAGAGCGATGGAGCAACAC

CAGACCTGGCCCTGGCAAGCCCCCCTGTGCAGAAGGCCGATAACGGGCCCAAGCAGTACGT

GTGGAGAAATATCCTGCTGTTCGCATATCTGCACGCCGCCGCCCTGTACGGCGGCTATCTG

TTTCTGACAAGCGGCAAGTGGCAGACGGACGTGTTCGCCTAGATCCTGTATGTGATGTCCG

GACTGGGAATCACAGCAGGAGCACACAGGCTGTGGGCACACAAGTCTTACAAGGCCAAGTG

GCCGCTGAAAGTGATCCTGATCATGTTTAACACCATCGCCTTTCAGGACGCAGCAATGGAT

TGGGCAAGGGACCACAGAATGCACCACAAGTATAGCGAGACAGACGCCGATCCTCACAATG

CCACCAGGGGCTTCTTTTTCTCCCACATCGGCTGGCTGCTGGTGCGCAAGCACCCAGATCT

GAAGGAGAAGGGCAAGGGCGTGGACATGAGCGATGTGCAGGGCGACCCCATCCTGCGGTTT

CAGAAGAAGTACTATCTGCTGCTGATGCCTCTGGCCTGCTTTGTGATGCCAACAGTGATCC

CCGTGTACTTCTGGGGCGAGACCTGGAACAATGCCTTTTTCGTGGCCGCCATGTTTAGATA

TGCCTTCATCCTGAACGTGACCTGGCTGGTGAATTCCGCCGCCCACAAGTGGGGCGATAAG

CCTTACGACAAGAGCATCAAGCCATCCGAGAACATGAGCGTGGCCATGTTTGCCCTGGGCG

AGGGCTTCCACAATTACCACCACACATTCCCCTGGGATTATAAGACCGCCGAGCTGGGCAA

CAATAAGCTGAACTTTACCACAACCTTCATCAACTTCTTCGCCAAGCTGGGCTGGGCCTAC

GACATGAAGACAGTGTCCGACGATATCGTGAAGAACAGGGTGAAGCGCACCGGCGATGGAT

CTCACCACCTGTGGGGATGGGGCGACAAGAACCAGAGCAAGGAGGAGATCGCCTCCGCCAT

CCGGATCAATCCTAAGGACGATTGA

SEQ ID NO: 97
DST180_amino_acid Z9 Desaturase encoded by SEQ ID NO: 96

MAPNISDDVNGVLFESDAATPDLALASPPVQKADNRPKQYVWRNILLFAYLHAAALYGGYL

FLTSAKWQTDVFAYILYVMSGLGITAGAHRLWAHKSYKAKWPLKVILIIFNTIAFQDAAMD

WARDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPDLKEKGKGLDMSDLQADPILRF

QKKYYLLLMPLACFVMPTVIPVYFWGETWNNAFFVAAMFRYAFILNVTWLVNSAAHKWGDK

PYDKSIKPSENMSVAMFALGEGFHNYHHTFPWDYKTAELGNNKLNFTTTFINFFAKLGWAY

DMKTVSDDIVKNRVKRTGDGSHHLWGWGDKNQSKEEIASAIRINPKDD

SEQ ID NO: 98
DST181_coding_sequence Z9 Desaturase

ATGGCCCCAAACATCTCTGAGGATGCCAATGGCGTGCTGTTTGAGAGCGATGCAGCAACAC

CAGACCTGGCCCTGGCAAGCCCACCTGTGCAGAAGGCAGACAACAGGCCCAAGGAGTACGT

GTGGAGAAATATCATCCTGTTTGCCTATCTGCACCTGGCCGCCCTGTACGGCGGCTATCTG

TTTCTGTTCAGCGCCAAGTGGCAGACAGACGTGTTCGCCTACATCCTGTATGTGATGTCCG

GACTGGGAATCACCGCAGGAGCACACAGACTGTGGGCACACAAGTCTTAGAAGGCCAAGTG

GCCCCTGAAAGTGATCCTGATCATCTTTAACACCATCGCCTTTCAGGACGCAGCAATGGAT

TGGGCAAGGGACCACAGAATGCACCACAAGTATAGCGAGACAGACGCCGATCCTCACAATG

CCACCAGGGGCTTCTTTTTCTCCCACATCGGCTGGCTGCTGGTGCGCAAGCACCCAGACCT

GAAGAAGAAGGGCAAGGGCCTGGACATGAGCGATCTGCTGAACGACCCCATCCTGAAGTTT

CAGAAGAAGTACTATCTGCTGCTGATGCCTCTGGCCTGCTTCGTGATGCCAACAATGATCC

CCGTGTACCTGTGGGGCGAGACATGGACCAATGCCTTTTTCGTGGCCGCCATGTTTCGGTA

TGCCTTCATCCTGAACGTGACCTGGCTGGTGAATTCCGCCGCCCACAAGTGGGGCGATAAG

CCTTAGGACAAGAGCATCAAGCCATCCGAGAACCTGTCTGTGGCCATGTTTGCCCTGGGCG

AGGGCTTCCACAATTACCACCACACATTCCCCTGGGATTATAAGACCGCCGAGCTGGGCAA

CCAGAAGCTGAACTTCACCACAACCTTCATCAACTTTTTCGCCAAGCTGGGCTGGGCCTAC

GACATGAAGACAGTGTCCGACGATATCGTGAAGAATAGGGTGAAGCGCACCGGCGATGGAT

CTCACCACCTGTGGGGATGGGGCGACAAGAACCAGAGCAAGGAGGAGATCGCCTCCGCCAT

CCGGATCAATCCTAAGGACGATTGA

SEQ ID NO: 99
DST181_amino_acid Z9 Desaturase encoded by SEQ ID NO: 98

MAPNISEDANGVLFESDAATPDLALASPPVQKADNRPKQYVWRNIILFAYLHLAALYGGYL

FLFSAKWQTDVFAYILYVMSGLGITAGAHRLWAHKSYKAKWPLKVILIIFNTIAFQDAAMD

WARDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPDLKKKGKGLDMSDLLMDPILKF

QKKYYLLLMPLACFVMPTMIPVYLWGETWTNAFFVAAMFRYAFILNVTWLVNSAAHKWGDK

PYDKSIKPSENLSVAMFALGEGFHNYHHTFPWDYKTAELGNQKLNFTTTFINFFAKLGWAY

DMKTVSDDIVKNRVKRTGDGSHHLWGWGDKNQSKEEIASAIRINPKDD

SEQ ID NO: 100
DST183_coding_sequence Z9 Desaturase

ATGGCCCCAAACATCAGCGAGGATGTGAATGGCGTGCTGTTCGAGTCCGATGCCGCCACAC

CAGACCTGGCCCTGTCTACCCCACCTGTGCAGAAGGCAGACAACAGGCCCAAGCAGCTGGT

GTGGAGAAATATCCTGCTGTTTGCATACCTGCACCTGGCAGCACAGTACGGAGGCTATCTG

TTTCTGTTCTCTGCCAAGTGGCAGACAGATATCTTCGCCTACATCCTGTATGTGATCAGCG

GACTGGGAATCACCGCAGGAGCACACCGGCTGTGGGCCCACAAGTCCTACAAGGCCAAGTG

GCCTCTGAGAGTGATCCTGGTCATCTTCAACACCGTGGCCTTTCAGGACGCAGCAATGGAT

TGGGCAAGGGACCACAGAATGCACCACAAGTATTCTGAGACAGACGCCGATCCTCACAATG

CCACCAGGGGCTTCTTTTTCAGCCACATCGGCTGGCTGCTGGTGCGCAAGCACCCAGATCT

GAAGGAGAAGGGCAAGGGCCTGGACATGAGCGATCTGCTGGCCGACCCCATCCTGAGGTTT

CAGAAGAAGTACTATCTGATCCTGATGCCTCTGGCCTGCTTTGTGATGCCAACAGTGATCC

CCGTGTACTTCTGGGGCGAGACATGGACCAACGCCTTTTTCGTGGCCGCCATGTTTCGCTA

TGCCTTCATCCTGAACGTGACCTGGCTGGTGAATTCTGCCGCCCACAAGTGGGGCGATAAG

CCTTAGGACAAGAGCATCAAGCCATCCGAGAACCTGTCTGTGGCCATGTTTGCCCTGGGCG

AGGGCTTCCACAATTACCACCACACATTCCCCTGGGACTATAAGACCGCCGAGCTGGGCAA

CAATAAGCTGAACTTTACCACAACCTTCATCAACTTCTTCGCCAAGATCGGCTGGGCCTAT

GATCTGAAGACAGTGTCCGACGATATCGTGAAGAATAGGGTGAAGAGGACCGGCGACGGAA

GCCACCACCTGTGGGGCTGGGGCGATGAGAACCAGTCCAAGGAGGAGATCGACGCCGCCAT

CCGGATCAATCCTAAGGACGATTGA

SEQ ID NO: 101
DST183_amino_acid Z9 Desatursae encoded by SEQ ID NO: 100

MAPNISEDVNGVLFESDAATPDLALSTPPVQKADNRPKQLVWRNILLFAYLHLAAQYGGYL

FLFSAKWQTDIFAYILYVISGLGITAGAHRLWAHKSYKAKWPLRVILVIFMTVAFQDAAMD

WARDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPDLKEKGKGLDMSDLLADPILRF

QKKYYLILMPLACFVMPTVIPVYFWGETWTNAFfVAAMFRYAFILNVTWLVNSAAHKWGDK

PYDKSIKPSENLSVAMFALGEGFHNYHHTFPWDYKTAELGNNKLNFTTTFINFFAKIGWAY

DLKTVSDDIVKNRVKRTGDGSHHLWGWGDENQSKEEIDAAIRINPKDD

SEQ ID NO: 102
DST189_coding_sequence Z9 Desaturase

ATGGCCCCTAACGTGACCGAGGAGAATGGCGTGCTGTTCGAGTCTGATGCAGCAACACCTG

ACCTGGCCCTGGCAAGAGAGCCAGTGGAGCAGGCAGATAGCTCCCCACGGGTGTACGTGTG

GAGAAACATCATCCTGTTTGCCTATCTGCACATCGCCGCCGTGTACGGCGGCTATCTGTTT

CTGTTCTCCGCCAAGTGGCAGACCGACATCTTCGCCTACCTGCTGTATGTGGCCTCTGGAC

TGGGAATCACAGCAGGAGCACACAGGCTGTGGGCCCACAAGAGCTACAAGGCCAAGTGGCC

CCTGAGACTGATCCTGACCATCTTTAACACCACAGCCTTTCAGGACAGCGCCATCGATTGG

GCCCGGGACCACAGAATGCACCACAAGTATTCCGAGACCGACGCCGATCCCCACAATGCCA

CAAGGGGCTTCTTTTTCTCCCACATCGGCTGGCTGCTGGTGAGGAAGCACCCTGAGCTGAA

GCGCAAGGGCAAGGGCCTGGACCTGTCTGATCTGTACGCCGATCCTATCCTGCGCTTTCAG

AAGAAGTACTATCTGATCCTGATGCCACTGGCCTGCTTCATCCTGCCCACCGTGATCCCCG

TGTACCTGTGGAACGAGACATGGAGCAATGCCTTTTTCGTGGCCGCCCTGTTTCGGTATAC

CTTCATCCTGAACGTGACATGGCTGCTGAATTCCGCCGCCCACAAGTGGGGCGATAAGCCA

TACGACAAGTCCATCAAGCCCTCTGAGAACCTGTCTGTGAGCCTGTTTGCCTTCGGCGAGG

GCTTTCACAATTACCACCACACCTTCCCATGGGATTATAAGACAGCCGAGCTGGGCAACCA

CCGGCTGAACTTCACCACAAAGTTCATCAACTTTTTCGCCAAGATCGGCTGGGCCTATGAT

ATGAAGACCGTGTCTCACGAGATCGTGCAGCAGAGGGTGAAGAGGACAGGCGACGGAAGCC

ACCACCTGTGGGGATGGGGCGACAAGGATCACGCACAGGAGGAGATCGACGCCGCCATCAG

AATCAATCCCAAGGACGATTGA

SEQ ID NO: 103
DST189_amino_acid Z9 Desaturase encoded by SEQ ID NO: 102

MAPNVTEENGVLFESDAATPDLALAREPVQQADSSPRVYVWRNIILFAYLHIAAVYGGYLF

LFSAKWQTDIFAYLLYVASGLGITAGAHRLWAHKSYKAKWPLRLILTIFNTTAFQDSAIDW

ARDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPELKRKGKGLDLSDLYADPILRFQ

KKYYLILMPLACFILPTVIPVYLWNETWSNAFFVAALFRYTFILNVTWLVNSAAHKWGDKP

YDKSIKPSENLSVSLFAFGEGFHNYHHTFPWDYKTAELGNHRLNFTTKFINFFAKIGWAYD

MKTVSHEIVQQRVKRTGDGSHHLWGWGDKDHAQEEIDAAIRINPKDD

SEQ ID NO: 104
DST192_coding_sequence Z9 Desaturase

ATGGATTTTCTGAACGAGATCGACAATTGCCCCGAGCGGCTGAGAAAGCCAGAGAAGATGG

CCCCCAACGTGACCGAGGAGAATGGCGTGCTGTTCGAGTCCGATGCAGCAACCCCAGACCT

GGCCCTGGCAAGGACACCTGTGGAGCAGGCCGACGATTCTCCAAGGATCTACGTGTGGCGC

AACATCATCCTGTTTGCCTATCTGCACCTGGCCGCCATCTACGGCGGCTATCTGTTTCTGT

TCTCCGCCAAGTGGCAGACCGATATCTTCGCCTACCTGCTGTATGTGGCATCTGGACTGGG

AATCACAGCAGGAGCACACAGGCTGTGGGCACACAAGAGCTACAAGGCCAAGTGGCCTCTG

CGCCTGATCCTGACCATCTTTAACACAATCGCCTTTCAGGACAGCGCCATCGATTGGGCCA

GGGACCACCGCATGCACCACAAGTATTCCGAGACCGACGCCGATCCACACAATGCCACACG

GGGCTTCTTTTTCTCTCACATCGGATGGCTGCTGGTGCGGAAGCACCCAGAGCTGAAGAGA

AAGGGCAAGGGCCTGGACCTGTCTGATCTGTACAGCGATCCCATCCTGAGATTTCAGAAGA

AGTACTATATGATCCTGATGCCTCTGGCCTGTTTCATCCTGCCCACCGTGATCCCCGTGTA

TATGTGGAACGAGACATGGAGCAATGCCTTTTTCGTGGCCGCCCTGTTTAGGTATACCTTC

ATCCTGAACGTGACATGGCTGGTGAATTCCGCCGCCCACAAGTGGGGCGATAAGCCTTACG

ACAAGTCCATCAAGCCATCTGAGAACATGAGCGTGTCCCTGTTTGCCTTCGGCGAGGGCTT

TCACAATTACCACCACACCTTCCCTTGGGACTATAAGACAGCCGAGCTGGGCAACCACCGG

CTGAACTTCACCACAAAGTTCATCAACTTCTTCGCCAAGATCGGCTGGGCCTATGATATGA

AGACCGTGTCTCAGGAGATCGTGCAGCAGCGGGTGAAGAGAACACGCGACGGAAGCCACCA

CCTGTGGGGATGGGGCGACAAGGATCACGCACAGGAGGAGATCAACGCCGCCATCCGCATC

AATCCAAAGGACGATTGA

SEQ ID NO: 105
DST192_amino_acid Z9 Desaturase encoded by SEQ ID NO: 104

MDFLNEIDNCPERlRKPEKMAPNVTeENGVLFESDAATPDLALARTPVEQADDSPRIYVWR

NIILFAYLHLAAIYGGYLFLFSAKWQTDIFAYLLYVASGLGITAGAHRLWAHKSYKAKWPL

RLILTIFNTIAFQDSAIDWARDHRMHHKYSETDADPHNATRGFFFSHIGWLLVRKHPELKR

KGKGLDLSDLYSDPILRFQKKYYMILMPLACFILPTVIPVYMWNETWSNAFFVAALFRYTF

ILNVTWLVNSAAHKWGDKPYDKSIKPSENMSVSLFAFGEGFHNYHHTFPWDYKTAELGNHR

LNFTTKFINFFAKIGWAYDMKTVSQEIVQQRVKRTGDGSHHLWGWGDKDHAQEEINAAIRI

NPKDD

The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood there from as modifications will be obvious to those skilled in the art.

While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. The current application hereby incorporates by reference each of the following in its entirety: U.S. Provisional Application Ser. No. 62/257,054, filed Nov. 18, 2015, U.S. Provisional Application Ser. No. 62/351,605, filed Jun. 17, 2016, and PCT application no. PCT/US2016/062852, filed Nov. 18, 2016.

However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Further Embodiments of the Invention

Other subject matter contemplated by the present disclosure is set out in the following numbered embodiments:

1. A recombinant Yarrowia lipolytica microorganism capable of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the recombinant Yarrowia lipolytica microorganism comprises:

- (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; and
- (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having at least 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.
  
  2. The recombinant Yarrowia lipolytica microorganism of embodiment 1, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C₆-C₂₄fatty alcohol.
  
  3. The recombinant Yarrowia lipolytica microorganism of embodiments 1 or 2, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following:
  
  (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6);
  
  (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7);
  
  (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1);
  
  (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and
  
  (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
  
  4. The recombinant Yarrowia lipolytica microorganism of embodiments 1 or 2, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion of one or more endogenous enzyme selected from the following:
  
  (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6);
  
  (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7);
  
  (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1);
  
  (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and
  
  (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
  
  5. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-4, wherein the fatty acyl desaturase catalyzes the conversion of a saturated fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from Z9-14:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, Z9-16:Acyl-CoA, and Z11-16:Acyl-CoA.
  
  6. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-5, wherein the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.
  
  7. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-6, wherein the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde.
  
  8. The recombinant Yarrowia lipolytica microorganism of embodiment 7, wherein the alcohol dehydrogenase is selected from Table 3a.
  
  9. The recombinant Yarrowia lipolytica microorganism of embodiments 7 or 8, wherein the C₆-C₂₄fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
  
  10. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-9, wherein the recombinant Yarrowia lipolytica microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate.
  
  11. The recombinant Yarrowia lipolytica microorganism of embodiment 10, wherein the acetyl transferase is selected from Table 5d.
  
  12. The recombinant Yarrowia lipolytica microorganism of embodiments 10 or 11, wherein the C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, and Z13-18:Ac.
  
  13. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-12, wherein the recombinant Yarrowia lipolytica microorganism further comprises:
  
  at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde; and
  
  at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate.
  
  14. The recombinant Yarrowia lipolytica microorganism of embodiment 13, wherein the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde and C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
  
  15. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-14, wherein the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66 and 67.
  
  16. The recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-15, wherein the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from an Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or Trichoplusia ni derived desaturase.
  
  17. A method of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating the recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-16 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol.
  
  18. The method of embodiment 17, wherein the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.
  
  19. The method of embodiments 17 or 18, wherein the recombinant Yarrowia lipolytica microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following:
  
  (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX1), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6);
  
  (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH), YALI0D25630g (ADH1), YALI0E17787g (ADH2), YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7);
  
  (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1);
  
  (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0E1320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B13838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8), YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK11) and YALI0A20130g (Alk12); and
  
  (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
  
  20. The method of embodiments 17 or 18, further comprising a step of recovering the mono- or poly-unsaturated C₆-C₂₄fatty alcohol.
  
  21. The method of embodiment 20, wherein said recovery step comprises distillation.
  
  22. The method of embodiment 20, wherein said recovery step comprises membrane-based separation.
  
  23. A method of producing a mono- or poly-unsaturated C₆-C₂₄fatty aldehyde from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating the recombinant Yarrowia lipolytica microorganism of any one of embodiment 1-16 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde.
  
  24. The method of embodiment 23, wherein the C₆-C₂₄fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
  
  25. The method of embodiments 23 or 24, further comprising a step of recovering the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde.
  
  26. The method of embodiment 25, wherein said recovery step comprises distillation.
  
  27. The method of embodiment 25, wherein said recovery step comprises membrane-based separation.
  
  28. A method of producing a mono- or poly-unsaturated C₆-C₂₄fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating the recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-16 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C₆-C₂₄fatty acetate.
  
  29. The method of embodiment 28, wherein the C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, and Z13-18:Ac.
  
  30. The method of embodiment 28, further comprising a step of recovering the mono- or poly-unsaturated C₆-C₂₄fatty acetate.
  
  31. The method of embodiment 28, wherein said recovery step comprises distillation.
  
  32. The method of embodiment 28, wherein said recovery step comprises membrane-based separation.
  
  33. A method of producing a mono- or poly-unsaturated C₆-C₂₄fatty aldehyde and C₆-C₂₄fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating the recombinant Yarrowia lipolytica microorganism of any one of embodiments 1-16 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde and C₆-C₂₄fatty acetate.
  
  34. The method of embodiment 33, wherein the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde and C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z1Z13-16:Ald and Z13-18:Ald.
  
  35. A method of engineering a Yarrowia lipolytica microorganism that is capable of producing a mono- or poly-unsaturated C₆-C₂₄fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the method comprises introducing into a Yarrowia lipolytica microorganism the following:
- (a) at least one nucleic acid molecule encoding a fatty acyl desaturase having at least 95% sequence identity to a fatty acyl desaturase selected from the group consisting of SEQ ID NOs: 39, 54, 60, 62, 78, 79, 80, 95, 97, 99, 101, 103, and 105 that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA; and
- (b) at least one nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase having at least 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of SEQ ID NOs: 41-48, 57, 73, 75 and 77 that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated C₆-C₂₄fatty alcohol.
  
  36. The method of embodiment 35, wherein the method further comprises introducing into the Yarrowia lipolytica microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated C₆-C₂₄fatty alcohol.
  
  37. The method of embodiment 35 or 36, wherein the Yarrowia lipolytica microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.
  
  38. The method of any one of embodiments 35-37, wherein the method further comprises introducing into the Yarrowia lipolytica microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from the following:
  
  (i) one or more acyl-CoA oxidase selected from the group consisting of YALI0E32835g (POX), YALI0F10857g (POX2), YALI0D24750g (POX3), YALI0E27654g (POX4), YALI0C23859g (POX5), YALI0E06567g (POX6);
  
  (ii) one or more (fatty) alcohol dehydrogenase selected from the group consisting of YALI0F09603g (FADH). YALI0D25630g (ADH1), YALI0E17787g (ADH2). YALI0A16379g (ADH3), YALI0E15818g (ADH4), YALI0D02167g (ADH5), YALI0A15147g (ADH6), YALI0E07766g (ADH7);
  
  (iii) a (fatty) alcohol oxidase YALI0B14014g (FAO1);
  
  (iv) one or more cytochrome P450 enzyme selected from the group consisting of YALI0E25982g (ALK1), YALI0F01320g (ALK2), YALI0E23474g (ALK3), YALI0B13816g (ALK4), YALI0B3838g (ALK5), YALI0B01848g (ALK6), YALI0A15488g (ALK7), (YALI0C12122g (ALK8).YALI0B06248g (ALK9), YALI0B20702g (ALK10), YALI0C10054g (ALK1) and YALI0A20130g (Alk12), and
  
  (v) one or more diacylglycerol acyltransferase selected from the group consisting of YALI0E32791g (DGA1) and YALI0D07986g (DGA2).
  
  39. The method of any one of embodiments 35-38, wherein the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from Z9-14:Acyl-CoA, Z11-14:Acyl-CoA, E11-14:Acyl-CoA, Z9-16:Acyl-CoA, and Z11-16:Acyl-CoA.
  
  40. The method of any one of embodiments 35-39, wherein the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is selected from the group consisting of Z9-14:OH, Z11-14:OH, E11-14:OH, Z9-16:OH, Z11-16:OH, Z11Z13-16:OH, and Z13-18:OH.
  
  41. The method of any one of embodiments 35-40, wherein the method further comprises introducing into or expressing in the recombinant Yarrowia lipolytica microorganism at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde.
  
  42. The method of embodiment 41, wherein the alcohol dehydrogenase is selected from Table 3a.
  
  43. The method of embodiment 41, wherein the C₆-C₂₄fatty aldehyde is selected from the group consisting of Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
  
  44. The method of any one of embodiment 35-43, wherein the method further comprises introducing into or expressing in the recombinant Yarrowia lipolytica microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate.
  
  45. The method of embodiment 44, wherein the acetyl transferase is selected from Table 5d.
  
  46. The method of embodiment 44, wherein the C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, and Z13-18:Ac.
  
  47. The method of any one of embodiments 35-46, wherein the method further comprises introducing into or expressing in the recombinant Yarrowia lipolytica microorganism:
  
  at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty aldehyde; and
  
  at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty alcohol into a corresponding C₆-C₂₄fatty acetate.
  
  48. The method of any one of embodiments 35-47, wherein the mono- or poly-unsaturated C₆-C₂₄fatty aldehyde and C₆-C₂₄fatty acetate is selected from the group consisting of Z9-14:Ac, Z11-14:Ac. E11-14:Ac, Z9-16:Ac, Z11-16:Ac, Z11Z13-16:Ac, Z13-18:Ac, Z9-14:Ald, Z11-14:Ald, E11-14:Ald, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald and Z13-18:Ald.
  
  49. The method of any one of embodiments 35-48, wherein the fatty acyl desaturase does not comprise a fatty acyl desaturase comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 65, 66 and 67.
  
  50. The method of any one of embodiments 35-49, wherein the fatty acyl desaturase does not comprise a fatty acyl desaturase selected from an Amyelois transitella, Spodoptera littoralis, Agrotis segetum, or Trichoplusia ni derived desaturase.
  
  51. The method of any one of embodiments 17-22, wherein the mono- or poly-unsaturated C₆-C₂₄fatty alcohol is converted into a corresponding C₆-C₂₄fatty aldehyde using chemical methods.
  
  53. The method of any one of embodiments 17-22, wherein the mono- or poly-unsaturated C₆-C₂s fatty alcohol is converted into a corresponding C₆-C₂₄fatty acetate using chemical methods.
  
  54. The method of embodiment 53, wherein the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N, N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated C₆-C₂₄fatty alcohol to the corresponding C₆-C₂₄fatty acetate.
  
  55. A recombinant microorganism capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the recombinant microorganism comprises:
- (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA;
- (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C₄-C₂₂fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA substrate in that cycle; and
- (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C₁₈fatty alcohol.
  
  56. The recombinant microorganism of embodiment 55, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C₁₈fatty acyl-CoA.
  
  57. The recombinant microorganism of any one of embodiments 55-56, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C₁₈fatty acyl-CoA, and wherein the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT).
  
  58. The recombinant microorganism of any one of embodiments 55-57, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C₁₈fatty acyl-CoA, and wherein the acyltransferase is selected from Table 5b.
  
  59. The recombinant microorganism of any one of embodiments 55-58, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates.
  
  60. The recombinant microorganism of any one of embodiments 55-59, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates, and wherein the acylglycerol lipase is selected from Table 5c.
  
  61. The recombinant microorganism of any one of embodiments 55-60, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzymes that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ≤C₁₈fatty alcohol.
  
  62. The recombinant microorganism of any one of embodiments 55-61, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from:
  
  (i) one or more acyl-CoA oxidase:
  
  (ii) one or more acyltransferase;
  
  (iii) one or more acylglycerol lipase and/or sterol ester esterase:
  
  (iv) one or more (fatty) alcohol dehydrogenase:
  
  (v) one or more (fatty) alcohol oxidase; and
  
  (vi) one or more cytochrome P450 monooxygenase.
  
  63. The recombinant microorganism of any one of embodiments 55-62, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g), Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g). Y. lipolytica POX5(YALI0C23859g). Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W); Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).
  
  64. The recombinant microorganism of any one of embodiments 55-63, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F19514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g. S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250. Candida CaO19.7881, Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.
  
  65. The recombinant microorganism of any one of embodiments 55-64, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138. Candida W5Q_03398, Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185. Candida CaO19.4864. Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854. Candida CTRG_05049, Candida CaO19.1887, Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.
  
  66. The method of any one of embodiments 55-65, wherein the recombinant microorganism comprises a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6). Y. lipolytica YALI10A5488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK2).
  
  67. The recombinant microorganism of any one of embodiments 55-66, wherein the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella. Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase.
  
  68. The recombinant microorganism of any one of embodiments 55-67, wherein the fatty acyl desaturase has at least 95% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, 78-80 and GenBank Accession nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and A1M40221.1.
  
  69. The recombinant microorganism of any one of embodiments 55-68, wherein the acyl-CoA oxidase is selected from Table 5a.
  
  70. The recombinant microorganism of any one of embodiments 55-69, wherein the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase.
  
  71. The recombinant microorganism of any one of embodiments 55-70, wherein the fatty alcohol forming fatty acyl reductase has at least 95% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82.
  
  72. The recombinant microorganism of any one of embodiments 55-71, wherein the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA, E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z15-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA, E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E4-16:Acyl-CoA, Z3E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z11Z13-16:Acyl-CoA, and Z13Z15-18:Acyl-CoA.
  
  73. The recombinant microorganism of any one of embodiments 55-72, wherein the mono- or poly-unsaturated ≤C₁₈fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH. Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH, and Z13-18:OH.
  
  74. The recombinant microorganism of any one of embodiments 55-73, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde.
  
  75. The recombinant microorganism of embodiment 74, wherein the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A117N158, and Nostoc punctiforme YP_001865324.
  
  76. The recombinant microorganism of any one of embodiments 55-75, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde.
  
  77. The recombinant microorganism of any one of embodiments 55-76, wherein the ≤C₁₈fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
  
  78. The recombinant microorganism of any one of embodiments 55-77, wherein the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty acetate.
  
  79. The recombinant microorganism of embodiment 78, wherein the acetyl transferase is selected from Table 5d.
  
  80. The recombinant microorganism of embodiment 78, wherein the ≤C₁₈fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac. E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, and Z11-16:Ac.
  
  81. The recombinant microorganism of any one of embodiments 55-80, wherein the recombinant microorganism further comprises:
  
  at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase, and an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde; and
  
  at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty acetate.
  
  82. The recombinant microorganism of embodiment 81, wherein the mono- or poly-unsaturated ≤C₁₈fatty aldehyde and ≤C₁₈fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z11-14:Ac, Z11-16:Ac, Z9-16:Ac, Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
  
  83. The recombinant microorganism of any one of embodiments 55-82, wherein the recombinant microorganism is a yeast selected from the group consisting of Yarrowia liptolytica, Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Candida viswanathii.
  
  84. A method of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating the recombinant microorganism of any one of embodiment 55-83 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ≤C₁₈fatty alcohol.
  
  85. The method of embodiment 84, wherein the mono- or poly-unsaturated ≤C₁₈fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH. E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH, Z9-16:OH, and Z13-18:OH.
  
  86. The method of any one of embodiments 84-85, further comprising a step of recovering the mono- or poly-unsaturated ≤C₁₈fatty alcohol.
  
  87. The method of embodiment 86, wherein said recovery step comprises distillation.
  
  88. The method of embodiment 86, wherein said recovery step comprises membrane-based separation.
  
  89. A method of engineering a microorganism that is capable of producing a mono- or poly-unsaturated ≤C₁₈fatty alcohol from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, wherein the method comprises introducing into a microorganism the following:
- (a) at least one exogenous nucleic acid molecule encoding a fatty acyl desaturase that catalyzes the conversion of a saturated C₆-C₂₄fatty acyl-CoA to a corresponding mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA;
- (b) at least one exogenous nucleic acid molecule encoding an acyl-CoA oxidase that catalyzes the conversion of the mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA from (a) into a mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA after one or more successive cycle of acyl-CoA oxidase activity, with a given cycle producing a mono- or poly-unsaturated C₄-C₂₂fatty acyl-CoA intermediate with a two carbon truncation relative to a starting mono- or poly-unsaturated C₆-C₂₄fatty acyl-CoA substrate in that cycle; and
- (c) at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty acyl reductase that catalyzes the conversion of the mono- or poly-unsaturated ≤C₁₈fatty acyl-CoA from (b) into the corresponding mono- or poly-unsaturated ≤C₁₈fatty alcohol.
  
  90. The method of embodiment 89, wherein the microorganism is MATA ura3-302::SUC2 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δfadh Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1::URA3.
  
  91. The method of any one of embodiments 89-90, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C₁₈fatty acyl-CoA.
  
  92. The method of any one of embodiments 89-91, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C₁₈fatty acyl-CoA, and wherein the acyltransferase is selected from the group consisting of glycerol-3-phosphate acyl transferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), glycerolphospholipid acyltransferase (GPLAT) and diacylglycerol acyltransferases (DGAT).
  
  93. The method of any one of embodiments 89-92, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acyltransferase that preferably stores ≤C₁₈fatty acyl-CoA, and wherein the acyltransferase is selected from Table 5b.
  
  94. The method of any one of embodiments 89-93, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates.
  
  95. The method of any one of embodiments 89-94, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acylglycerol lipase that preferably hydrolyzes ester bonds of >C16, of >C14, of >C12 or of >C10 acylglycerol substrates, and wherein the acylglycerol lipase is selected from Table 5c.
  
  96. The method of any one of embodiments 89-95, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme that catalyzes a reaction in a pathway that competes with the biosynthesis pathway for the production of a mono- or poly-unsaturated ≤C₁₈fatty alcohol.
  
  97. The method of any one of embodiments 89-96, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous enzyme selected from:
  
  (i) one or more acyl-CoA oxidase;
  
  (ii) one or more acyltransferase;
  
  (iii) one or more acylglycerol lipase and/or sterol ester esterase:
  
  (iv) one or more (fatty) alcohol dehydrogenase;
  
  (v) one or more (fatty) alcohol oxidase; and
  
  (vi) one or more cytochrome P450 monooxygenase.
  
  98. The method of any one of embodiments 89-97, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyl-CoA oxidase enzyme selected from the group consisting of Y. lipolytica POX1(YALI0E32835g), Y. lipolytica POX2(YALI0F10857g). Y. lipolytica POX3(YALI0D24750g), Y. lipolytica POX4(YALI0E27654g), Y. lipolytica POX5(YALI0C23859g), Y. lipolytica POX6(YALI0E06567g); S. cerevisiae POX1(YGL205W)S Candida POX2 (CaO19.1655, CaO19.9224, CTRG_02374, M18259), Candida POX4 (CaO19.1652, CaO19.9221, CTRG_02377, M12160), and Candida POX5 (CaO19.5723, CaO19.13146, CTRG_02721, M12161).
  
  99. The method of any one of embodiments 89-98, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acyltransferase enzyme selected from the group consisting of Y. lipolytica YALI0C00209g, Y. lipolytica YALI0E18964g, Y. lipolytica YALI0F9514g, Y. lipolytica YALI0C14014g, Y. lipolytica YALI0E16797g, Y. lipolytica YALI0E32769g, and Y. lipolytica YALI0D07986g, S. cerevisiae YBL011w, S. cerevisiae YDL052c, S. cerevisiae YOR175C, S. cerevisiae YPR139C, S. cerevisiae YNR008w, and S. cerevisiae YOR245c, and Candida I503_02577, Candida CTRG_02630, Candida CaO19.250, Candida CaO19.7881. Candida CTRG_02437, Candida CaO19.1881, Candida CaO19.9437, Candida CTRG_01687, Candida CaO19.1043, Candida CaO19.8645, Candida CTRG_04750, Candida CaO19.13439, Candida CTRG_04390, Candida CaO19.6941, Candida CaO19.14203, and Candida CTRG_06209.
  
  100. The method of any one of embodiments 89-99, wherein the method further comprises introducing into the microorganism one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous acylglycerol lipase and/or sterol ester esterase enzyme selected from the group consisting of Y. lipolytica YALI0E32035g, Y. lipolytica YALI0D17534g, Y. lipolytica YALI0F10010g, Y. lipolytica YALI0C14520g, and Y. lipolytica YALI0E00528g, S. cerevisiae YKL140w, S. cerevisiae YMR313c, S. cerevisiae YKR089c, S. cerevisiae YOR081c, S. cerevisiae YKL094W, S. cerevisiae YLL012W, and S. cerevisiae YLR020C, and Candida CaO19.2050, Candida CaO19.9598, Candida CTRG_01138, Candida W5Q_03398. Candida CTRG_00057, Candida CaO19.5426, Candida CaO19.12881, Candida CTRG_06185, Candida CaO19.4864, Candida CaO19.12328, Candida CTRG_03360, Candida CaO19.6501, Candida CaO19.13854, Candida CTRG_05049, Candida CaO19.1887. Candida CaO19.9443, Candida CTRG_01683, and Candida CTRG_04630.
  
  101. The method of any one of embodiments 89-100, wherein the method further comprises one or more modifications comprising a deletion, disruption, mutation, and/or reduction in the activity of one or more endogenous cytochrome P450 monooxygenases selected from the group consisting of Y. lipolytica YALI0E25982g (ALK1), Y. lipolytica YALI0F01320g (ALK2), Y. lipolytica YALI0E23474g (ALK3), Y. lipolytica YALI0B13816g (ALK4), Y. lipolytica YALI0B13838g (ALK5), Y. lipolytica YALI0B01848g (ALK6), Y. lipolytica YALI0A5488g (ALK7), Y. lipolytica YALI0C12122g (ALK8), Y. lipolytica YALI0B06248g (ALK9), Y. lipolytica YALI0B20702g (ALK10), Y. lipolytica YALI0C10054g (ALK11) and Y. lipolytica YALI0A20130g (ALK12).
  
  102. The method of any one of embodiments 89-101, wherein the fatty acyl desaturase is selected from an Argyrotaenia velutinana, Spodoptera litura, Sesamia inferens, Manduca sexta, Ostrinia nubilalis, Helicoverpa zea, Choristoneura rosaceana, Drosophila melanogaster, Spodoptera littoralis, Lampronia capitella, Amyelois transitella, Trichoplusia ni, Agrotis segetum, Ostrinia furnicalis, and Thalassiosira pseudonana derived fatty acyl desaturase.
  
  103. The method of any one of embodiments 89-102, wherein the fatty acyl desaturase has at least 95% sequence identity to a fatty acyl desaturase selected from the group consisting of: SEQ ID NOs: 39, 49-54, 58-63, and GenBank Accession nos. AF416738, AGH12217.1, AII21943.1, CAJ43430.2, AF441221, AAF81787.1, AF545481, AJ271414, AY362879, ABX71630.1, NP001299594.1, Q9N9Z8, ABX71630.1 and AIM40221.1.
  
  104. The method of any one of embodiments 89-103, wherein the acyl-CoA oxidase is selected from Table 5a.
  
  105. The method of any one of embodiments 89-104, wherein the fatty alcohol forming fatty acyl reductase is selected from an Agrotis segetum, Spodoptera exigua, Spodoptera littoralis, Euglena gracilis, Yponomeuta evonymellus and Helicoverpa armigera derived fatty alcohol forming fatty acyl reductase.
  
  106. The method of any one of embodiments 89-105, wherein the fatty alcohol forming fatty acyl reductase has at least 90% sequence identity to a fatty alcohol forming fatty acyl reductase selected from the group consisting of: SEQ ID NOs: 1-3, 32, 41-48, 55-57, 73, 75, 77 and 82.
  
  107. The method of any one of embodiments 89-106, wherein the fatty acyl desaturase catalyzes the conversion of a fatty acyl-CoA into a mono- or poly-unsaturated intermediate selected from E5-10:Acyl-CoA, E7-12:Acyl-CoA, E9-14:Acyl-CoA, E11-16:Acyl-CoA, E13-18:Acyl-CoA, Z7-12:Acyl-CoA, Z9-14:Acyl-CoA, Z11-16:Acyl-CoA, Z13-18:Acyl-CoA, Z8-12:Acyl-CoA, Z10-14:Acyl-CoA, Z12-16:Acyl-CoA, Z14-18:Acyl-CoA, Z7-10:Acyl-coA, Z9-12:Acyl-CoA, Z11-14:Acyl-CoA, Z13-16:Acyl-CoA, Z15-18:Acyl-CoA, E7-10:Acyl-CoA, E9-12:Acyl-CoA, E11-14:Acyl-CoA, E13-16:Acyl-CoA, E15-18:Acyl-CoA, E5Z7-12:Acyl-CoA, E7Z9-12:Acyl-CoA, E9Z11-14:Acyl-CoA, E11Z13-16:Acyl-CoA, E13Z15-18:Acyl-CoA, E6E8-10:Acyl-CoA, E8E10-12:Acyl-CoA, E10E12-14:Acyl-CoA, E12E14-16:Acyl-CoA, Z5E8-10:Acyl-CoA, Z7E10-12:Acyl-CoA, Z9E12-14:Acyl-CoA, Z11E14-16:Acyl-CoA, Z13E16-18:Acyl-CoA, Z3-10:Acyl-CoA, Z5-12:Acyl-CoA, Z7-14:Acyl-CoA, Z9-16:Acyl-CoA, Z11-18:Acyl-CoA, Z3Z5-10:Acyl-CoA, Z5Z7-12:Acyl-CoA, Z7Z9-14:Acyl-CoA, Z9Z11-16:Acyl-CoA, Z11Z13-16:Acyl-CoA, and Z3Z15-18:Acyl-CoA.
  
  108. The method of any one of embodiments 89-107, wherein the mono- or poly-unsaturated ≤C₁₈fatty alcohol is selected from the group consisting of E5-10:OH, Z8-12:OH, Z9-12:OH, Z11-14:OH, Z11-16:OH, E11-14:OH, E8E10-12:OH, E7Z9-12:OH, Z11Z13-16OH, Z9-14:OH. Z9-16:OH, and Z13-18:OH.
  
  109. The method of any one of embodiments 89-108, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde.
  
  110. The method of embodiment 109, wherein the aldehyde forming fatty acyl-CoA reductase is selected from the group consisting of Acinetobacter calcoaceticus A0A1C4HN78, A. calcoaceticus N9DA85, A. calcoaceticus R8XW24, A. calcoaceticus A0A1A0GGM5, A. calcoaceticus A0A117N158, and Nostoc punctiforme YP_001865324.
  
  111. The method of any one of embodiments 89-110, wherein the method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty aldehyde.
  
  112. The method of any one of embodiments 109-111, wherein the ≤C₁₈fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
  
  113. The method of any one of embodiments 89-112, wherein method further comprises introducing into the microorganism at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty acetate.
  
  114. The method of embodiment 113, wherein the acetyl transferase is selected from Table 5d.
  
  115. The method of any one of embodiment 113-114, wherein the ≤C₁₈fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, E11-14:Ac, Z9-16:Ac, Z11-14:Ac and Z11-16:Ac.
  
  116. The method of any one of embodiments 89-115, wherein the method further comprises introducing into the microorganism:
  
  at least one endogenous or exogenous nucleic acid molecule encoding an enzyme selected from an alcohol oxidase, an alcohol dehydrogenase, and an aldehyde forming fatty acyl-CoA reductase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈₁fatty aldehyde; and
  
  at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated ≤C₁₈fatty alcohol into a corresponding ≤C₁₈fatty acetate.
  
  117. The method of embodiment 116, wherein the mono- or poly-unsaturated ≤C₁₈fatty aldehyde and ≤C₁₈fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z11-16:Ac, Z9-16:Ald, Z9-16:Ac, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
  
  118. A method of producing a mono- or poly-unsaturated ≤C₁₈fatty aldehyde from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating the recombinant microorganism of any one of embodiments 74-76 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ≤C₁₈fatty aldehyde.
  
  119. The method of embodiment 118, wherein the ≤C₁₈fatty aldehyde is selected from the group consisting of Z9-16:Ald, Z11-16:Ald, Z11Z13-16:Ald, and Z13-18:Ald.
  
  120. The method of any one of embodiments 118-119, further comprising a step of recovering the mono- or poly-unsaturated ≤C₁₈fatty aldehyde.
  
  121. The method of embodiment 120, wherein said recovery step comprises distillation.
  
  122. The method of embodiment 120, wherein said recovery step comprises membrane-based separation.
  
  123. A method of producing a mono- or poly-unsaturated ≤C₁₈fatty acetate from an endogenous or exogenous source of saturated C₆-C₂₄fatty acid, comprising: cultivating the recombinant microorganism of any one of embodiments 78-80 in a culture medium containing a feedstock that provides a carbon source adequate for the production of the mono- or poly-unsaturated ≤C₁₈fatty acetate.
  
  124. The method of embodiment 123, wherein the mono- or poly-unsaturated ≤C₁₈fatty acetate is selected from the group consisting of E5-10:Ac, Z7-12:Ac, Z8-12:Ac, Z9-12:Ac, E7Z9-12:Ac, Z9-14:Ac, Z9E12-14:Ac, Z11-14:Ac, E11-14:Ac, Z9-16:Ac, and Z11-16:Ac.
  
  125. The method of any one of embodiments 123-124, further comprising a step of recovering the mono- or poly-unsaturated ≤C₁₈fatty acetate.
  
  126. The method of embodiment 125, wherein said recovery step comprises distillation.
  
  127. The method of embodiment 125, wherein said recovery step comprises membrane-based separation.
  
  128. The method of any one of embodiments 89-115, wherein the recombinant microorganism is a yeast selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida albicans, Candida viswanathii and Candida tropicalis.
  
  129. The method of any one of embodiments 89-115, wherein the mono- or poly-unsaturated ≤C₁₈fatty alcohol is converted into a corresponding ≤C₁₈fatty aldehyde using chemical methods.
  
  130. The method of embodiment 129, wherein the chemical methods are selected from TEMPO-bleach, TEMPO-copper-air, TEMPO-PhI(OAc)₂, Swern oxidation and noble metal-air.
  
  131. The method of any one of embodiments 89-115, wherein the mono- or poly-unsaturated ≤C₁₈fatty alcohol is converted into a corresponding ≤C₁₈fatty acetate using chemical methods.
  
  132. The method of embodiment 131, wherein the chemical methods utilize a chemical agent selected from the group consisting of acetyl chloride, acetic anhydride, butyryl chloride, butyric anhydride, propanoyl chloride and propionic anhydride in the presence of 4-N,N-dimethylaminopyridine (DMAP) or sodium acetate to esterify the mono- or poly-unsaturated ≤C₁₈fatty alcohol to the corresponding ≤C₁₈fatty acetate.

Number	Name	Date	Kind
4231947	Schrock	Nov 1980	A
4245131	Schrock	Jan 1981	A
4427595	Schrock	Jan 1984	A
4681956	Schrock	Jul 1987	A
4727215	Schrock	Feb 1988	A
5087710	Schrock et al.	Feb 1992	A
5124491	Fleckenstein et al.	Jun 1992	A
5142073	Schrock et al.	Aug 1992	A
5146033	Schrock et al.	Sep 1992	A
6121473	Schrock et al.	Sep 2000	A
6291742	Somerville et al.	Sep 2001	B1
6346652	Schrock et al.	Feb 2002	B1
7169959	Heck et al.	Jan 2007	B2
7700833	Renz et al.	Apr 2010	B2
8110093	Friedman et al.	Feb 2012	B2
8110670	Hu et al.	Feb 2012	B2
8268599	Schirmer et al.	Sep 2012	B2
8283143	Hu et al.	Oct 2012	B2
8535916	Del Cardayre et al.	Sep 2013	B2
8658404	Schirmer et al.	Feb 2014	B2
8987531	Grubbs et al.	Mar 2015	B2
9017984	Hu et al.	Apr 2015	B2
9068201	Hu et al.	Jun 2015	B2
9200299	Friedman et al.	Dec 2015	B2
9598706	Keasling et al.	Mar 2017	B2
9776179	Wampler et al.	Oct 2017	B2
10017455	Hu et al.	Jul 2018	B2
10093950	Gatter et al.	Oct 2018	B2
10308962	Leonard et al.	Jun 2019	B1
20060078973	Renz et al.	Apr 2006	A1
20070282148	Berlin et al.	Dec 2007	A1
20080009598	Herrmann et al.	Jan 2008	A1
20080119678	Hock et al.	May 2008	A1
20080207911	Herrmann et al.	Aug 2008	A1
20080221345	Winde et al.	Sep 2008	A1
20080275247	Kadyrov et al.	Nov 2008	A1
20100087644	Mauduit et al.	Apr 2010	A1
20100113795	Arlt et al.	May 2010	A1
20100170826	Friedman et al.	Jul 2010	A1
20100174068	Grela et al.	Jul 2010	A1
20100199548	Del Cardayre et al.	Aug 2010	A1
20100235934	Friedman et al.	Sep 2010	A1
20100242345	Keasling et al.	Sep 2010	A1
20100251601	Hu et al.	Oct 2010	A1
20110015430	Schrock et al.	Jan 2011	A1
20110282068	Herrmann et al.	Jan 2011	A1
20110040099	Kadyrov et al.	Feb 2011	A1
20110065915	Malcolmson et al.	Mar 2011	A1
20110077421	Schrock	Mar 2011	A1
20110097769	Del Cardayre et al.	Apr 2011	A1
20110237815	Hock et al.	Sep 2011	A1
20110256599	Hu et al.	Oct 2011	A1
20120123133	Berlin et al.	May 2012	A1
20120142979	Keasling et al.	Jun 2012	A1
20120264983	Hu et al.	Oct 2012	A1
20120302710	Hoveyda et al.	Nov 2012	A1
20120323000	Hoveyda et al.	Dec 2012	A1
20130079515	Grela et al.	Mar 2013	A1
20130116434	Schrock et al.	May 2013	A1
20130144060	Mauduit et al.	Jun 2013	A1
20130211096	Arlt et al.	Aug 2013	A1
20130245339	Keasling et al.	Sep 2013	A1
20130261312	Allen et al.	Oct 2013	A1
20130274482	Schrock et al.	Oct 2013	A1
20130281688	Di Biase et al.	Oct 2013	A1
20130281706	Hock et al.	Oct 2013	A1
20130296511	Ung et al.	Nov 2013	A1
20140171607	Grela et al.	Jun 2014	A1
20140330018	Czirok et al.	Nov 2014	A1
20140378637	Schrock et al.	Dec 2014	A1
20150018557	Nolan et al.	Jan 2015	A1
20150038723	Herrmann et al.	Feb 2015	A1
20150045558	Plenio et al.	Feb 2015	A1
20150125933	Groban et al.	May 2015	A1
20150166465	Chen et al.	Jun 2015	A1
20150275188	Hu et al.	Oct 2015	A1
20160046914	Hom et al.	Feb 2016	A1
20160076058	Friedman et al.	Mar 2016	A1
20160108436	Coelho et al.	Apr 2016	A1
20160222419	Stuart	Aug 2016	A1
20160304913	Gatter et al.	Oct 2016	A1
20170275651	Keasling et al.	Sep 2017	A1
20170327799	Hu et al.	Nov 2017	A1
20180162916	Borodina et al.	Jun 2018	A1
20180371510	Gatter et al.	Dec 2018	A1
20190031594	Hu et al.	Jan 2019	A1
20190136272	Otte et al.	May 2019	A1
20190338317	Leonard et al.	Nov 2019	A1
20190376094	Friedman et al.	Dec 2019	A1
20200017890	Sanchez-Riera et al.	Jan 2020	A1

Number	Date	Country
1656226	Aug 2005	CN
101490241	Jul 2009	CN
102264910	Nov 2011	CN
102807470	Dec 2012	CN
103842502	Jun 2014	CN
104370701	Feb 2015	CN
104781411	Jul 2015	CN
104968672	Oct 2015	CN
108138202	Jun 2018	CN
15174099	Jun 2015	EP
H10-506783	Jul 1998	JP
WO 1991009825	Jul 1991	WO
WO 1992019631	Nov 1992	WO
WO 2007075427	Jul 2007	WO
WO 2007136762	Nov 2007	WO
WO 2007140954	Dec 2007	WO
WO 2008066754	Jun 2008	WO
WO 2008100251	Aug 2008	WO
WO 2008113041	Sep 2008	WO
WO 2008119082	Oct 2008	WO
WO 2008147781	Dec 2008	WO
WO 2009126831	Oct 2009	WO
WO 2010037550	Apr 2010	WO
WO 2010144296	Dec 2010	WO
WO 2011040963	Apr 2011	WO
WO 2011069134	Jun 2011	WO
WO 2011091980	Aug 2011	WO
WO 2011097642	Aug 2011	WO
WO 2012087964	Jun 2012	WO
WO 2012167171	Dec 2012	WO
WO 2012168183	Dec 2012	WO
WO 2013135776	Mar 2013	WO
WO 2013070725	May 2013	WO
WO 2014001291	Jan 2014	WO
WO 2014008054	Jan 2014	WO
WO 2014067767	May 2014	WO
WO 2014134333	Sep 2014	WO
WO 2014139679	Sep 2014	WO
WO 2014155185	Oct 2014	WO
WO 2014169014	Oct 2014	WO
WO 2014172534	Oct 2014	WO
WO 2015003814	Jan 2015	WO
WO 2015003815	Jan 2015	WO
WO 2015042306	Mar 2015	WO
WO 2015077752	May 2015	WO
WO 2015086684	Jun 2015	WO
WO 2015171057	Nov 2015	WO
WO 2016099568	Jun 2016	WO
WO 2016159869	Oct 2016	WO
WO 2016207339	Dec 2016	WO
WO 2017087846	May 2017	WO
WO 2017214133	Dec 2017	WO
WO 2018213554	Nov 2018	WO

Microorganisms for the production of insect pheromones and related compounds

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